Patent Publication Number: US-2022235389-A1

Title: Design of Protein Glycosylation Sites by Rapid Expression and Characterization of N-Glycosyltransferases

Description:
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS 
     The present application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/545,760, filed on Aug. 15, 2017, the content of which is incorporated herein by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under HDTRA1-15-1-0052/P00001 awarded by the Defense Threat Reduction Agency. The government has certain rights in the invention. 
    
    
     BACKGROUND 
     The present invention generally relates to components, systems, and methods for glycoprotein protein synthesis. In particular, the present invention relates to identification of amino acid glycosylation tag motifs (“GlycTags”) for N-glycosyltransferases and their use in synthesizing glycoproteins and recombinant glycoproteins in cells, using purified enzymes, or in cell-free protein synthesis (CFPS). 
     Glycosylation modulates the pharmacokinetics and potency of protein therapeutics and vaccines. However, current methods cannot sufficiently explore the vast experimental landscapes required to accurately predict and design glycosylation sites for specific glycosyltransferases (GTs). Here, we used a systematic platform for Glycosylation Sequence Characterization and Optimization by Rapid Expression and Screening (GlycoSCORES) using Cell-Free Protein Synthesis (CFPS) and Self-Assembled Monolayers for Desorption Ionization Mass Spectrometry (SAMDI-MS) to produce five cytoplasmic N-linked glycosyltransferases (NGTs) in vitro and determine their peptide acceptor and sugar donor specificities at unprecedented depth and throughput with ˜3,000 unique peptides and ˜10,000 unique reaction conditions. We found that peptide selectivity data closely matched glycosylation trends on small sequon motifs (GlycTags) within heterologous proteins, including an Fc human antibody fragment, in vitro and in the cytoplasm of living cells. The data collected in this work allows for design of polypeptide sequences for efficient, site-specific modification with NGTs and the GlycoSCORES workflow provides a systematic tool to characterize other polypeptide glycosyltransferases, and site-specifically control glycosylation structures. 
     SUMMARY 
     Disclosed are components, systems, and methods for glycoprotein protein synthesis in vitro and in vivo. In particular, the present invention relates to components, systems, and methods for identifying amino acid glycosylation tag motifs (“GlycTags”) for N-glycosyltransferases. The amino acid sequence of a protein may be modified to include a GlycTag that has been identified by the disclosed components, systems, and methods. The modified amino acid sequence of the protein then may be expressed in vitro, for example in a cell-free protein synthesis (CFPS) system, or in vivo, for example in a recombinant prokaryotic cell, in the presence of the corresponding N-glycosyltransferase and a sugar donor for the N-glycosyltransferase, where the N-glycosyltransferase transfers the sugar to the corresponding GlycTag in the amino acid sequence of the expressed protein to prepare a glycosylated variant of the protein. 
     As such, the disclosed methods may include methods for synthesizing a glycoprotein and/or a recombinant glycoprotein, for example a recombinant glycoprotein variant of a target protein. The disclosed methods may comprise (a) expressing in vivo, for example in a prokaryotic cell or a eukaryotic cell, or in vitro, for example in a prokaryotic-based or a eukaryotic based cell-free protein synthesis (CFPS) reaction, a polypeptide comprising the amino acid sequence of a target protein which includes an amino acid motif or that has been modified to include a heterologous amino acid motif (i.e., a “GlycTag”) that is glycosylated by an N-glycosyltransferase. In some embodiment, the amino acid motif or heterologous amino acid motif comprises an amino acid sequence selected from SEQ ID NOs:1-1-549, for example where the amino acid sequence of the target protein has been modified to include an amino acid sequence selected from SEQ ID NOs:1-549. For example, the amino acid sequence of one of SEQ ID NOs: 1-549 may be present or inserted into the amino acid sequence of the target protein. Alternatively, the amino acid sequence of the target protein may be modified by replacing one or more amino acids such that an amino acid sequence selected from SEQ ID NOs: 1-549 is present in the modified amino acid sequence of the target protein. The disclosed methods for synthesizing a glycoprotein optionally also may comprise (b) expressing in vivo, for example in a prokaryotic cell or eukaryotic cell, or in vitro, for example in a prokaryotic-based or eukaryotic CFPS reaction, the N-glycosyltransferase which glycosylates the amino acid motif or heterologous amino acid motif; and optionally also may comprise (c) reacting the polypeptide and the N-glycosyltransferase in the presence of a sugar donor, wherein the N-glycosyltransferase glycosylates the amino acid motif or heterologous amino acid motif of the polypeptide with the sugar to synthesize the glycoprotein or recombinant glycoprotein. 
     In the disclosed methods for synthesizing a glycoprotein or recombinant glycoprotein, the amino acid motif or heterologous amino acid motif (i.e., a “GlycTag”) may comprise an amino acid sequence of one of SEQ ID NOs:1-549. In some embodiments, the amino acid motif or heterologous amino acid motif comprises a sequence X −2 -X −1 -N-X +1 -S/T-X +3 , wherein X −2  is selected from Gly, Asn, and Tyr; and/or X −1  is selected from Gly and Ala; and/or X +1  is selected from Trp, Val, His, Ala, and Be; and/or X +3  is selected from Thr, Met, and Phe. 
     In some embodiments of the disclosed methods for synthesizing a recombinant glycoprotein, a target protein whose amino acid sequence is modified to prepare a recombinant glycoprotein variant may be a eukaryotic protein. In other embodiments of the disclosed methods for synthesizing a recombinant glycoprotein, a target protein whose amino acid sequence is modified to prepare a recombinant glycoprotein variant may be a prokaryotic protein. 
     In the disclosed methods for synthesizing a glycoprotein or recombinant glycoprotein, the steps of the method may be performed in vivo, for example in a prokaryotic cell or a eukaryotic cell, or in vitro, for example in a prokaryotic-based or a eukaryotic-based CFPS reaction. In some embodiments, one or more steps of the methods for synthesizing a glycoprotein or recombinant glycoprotein may be performed in vitro, for example in a prokaryotic-based or a eukaryotic-based CFPS reaction, and one or more other steps of the methods for synthesizing a glycoprotein or recombinant glycoprotein may be performed in vivo, for example in a prokaryotic cell or a eukaryotic cell. In other embodiments, all steps of the methods for synthesizing a glycoprotein or recombinant glycoprotein are performed in vitro, for example in a prokaryotic-based or a eukaryotic-based CFPS reaction, or all steps of the methods for synthesizing a glycoprotein or recombinant glycoprotein are performed in vitro, for example in a prokaryotic-based or a eukaryotic-based CFPS reaction. Suitable prokaryotic-based CFPS reactions for the disclosed methods may include, but are not limited to, an  Escherichia coli -based CFPS reaction (i.e., where a lysate from recombinant  E. coli  is used in the CFPS reaction). Suitable eukaryotic-based CFPS reactions for the disclosed methods include, but are not limited to a  Saccharomyces - cerevisiae -based CFPS reaction (i.e., where a lysate from recombinant  Saccharomyces - cerevisiae  is used in the CFPS reaction). 
     In the disclosed methods for synthesizing a glycoprotein or recombinant glycoprotein, an N-glycosyltransferase typically is expressed and utilized to glycosylate a modified amino acid sequence of a target protein. In some embodiments, the N-glycosyltransferase is a prokaryotic N-glycosyltransferase. Suitable N-glycosyltransferases may include but are not limited to an N-glycosyltransferase from one of  Actinobacillus  spp.,  Escherichia  spp.,  Haemophilus  spp., or  Mannheimia  spp. In particular, suitable N-glycosyltransferases may include but are not limited to an N-glycosyltransferase from one of  Actinobacillus pleuropneumoniae, Escherichia coli, Haemophilus influenza, Mannheimia haemolytica , or  Haemophilus dureyi . In other embodiments, the N-glycosyltransferase is a eukaryotic N-glycosyltransferase. 
     Also disclosed are methods for synthesizing a glycoprotein or recombinant glycoprotein, the methods comprising: (a) expressing in a cell or in a cell-free protein synthesis (CFPS) reaction a polypeptide comprising the amino acid sequence of a target protein which includes naturally two or more different amino acid motifs that includes an asparagine that is glycosylated by two or more different N-glycosyltransferases or that has been modified to include two or more different heterologous amino acid motifs that includes an asparagine that is glycosylated by two or more different N-glycosyltransferases, the amino acid motifs or heterologous amino acid motifs optionally comprising an amino acid sequence selected from SEQ ID NOs:1-549; (b) expressing in one or more cells or in one or more CFPS reactions the two or more different N-glycosyltransferases, where the two or more different N-glycosyltransferases are expressed simultaneously in the same cell or CFPS reaction or sequentially in two or more different cells or two or more different CFPS reactions; and (c) reacting the polypeptide and the two or more different N-glycosyltransferases in the presence of two or more sugar donors which are the same or different, where the polypeptide is reacted with the two or more different N-glycosyltransferases simultaneously for example in the same cell or CFPS reaction, or sequentially for example in two or more different cells or two or more different CFPS reactions, and where the two or more different N-glycosyltransferases glycosylate the two or more different amino acid motifs or heterologous amino acid motifs of the polypeptide with the sugar of the two or more sugar donors to synthesize the recombinant glycoprotein. 
     Also disclosed herein are methods for selecting an amino acid motif that is glycosylated by an N-glycosyltransferase. The disclosed methods may include (a) reacting a library of peptides comprising different amino acid motifs with a recombinant N-glycosyltransferase in the presence of a sugar donor, where the N-glycosyltransferase glycosylates one or more of the amino acid motifs of the peptides; and; (b) detecting glycosylation of the peptides to select the amino acid motif that is glycosylated by the N-glycosyltransferase. 
     In particular, the disclosed methods for selecting an amino acid motif that is glycosylated by an N-glycosyltransferase may include: (a) reacting a library of peptides comprising different amino acid motifs with an N-glycosyltransferase in the presence of a sugar donor, wherein the N-glycosyltransferase glycosylates one or more of the different amino acid motifs of the peptides; (b) detecting glycosylation of the reacted peptides by immobilizing the reacted peptides on a substrate comprising self-assembled monolayers, and performing matrix-assisted laser desorption/ionization mass spectrometry of the immobilized reacted peptides to select the amino acid motif that is glycosylated by the N-glycosyltransferase. In some embodiments of the disclosed selection methods, the library of peptides comprise a C-terminal Cys, the self-assembled monolayers comprise free maleimides, and the C-terminal Cys of the peptides reacts with the free maleimides to form a bond (e.g., a C—S bond) and covalently immobilize the peptide. In other embodiments of the disclosed selection methods, the library of peptides comprise a C-terminal alkyne, the self-assembled monolayers comprise free azides, and the C-terminal alkyne of the peptides reacts with the free azides to form a bond (e.g., a triazole and in particular a 1,2,3-triazole) and covalently immobilize the peptide. In alternative embodiments of the disclosed selection methods, the library of peptides comprise a C-terminal azide, the self-assembled monolayers comprise free alkynes, and the C-terminal azide of the peptides reacts with the free alkynes to form a bond (e.g., a triazole and in particular a 1,2,3-triazole) and covalently immobilize the peptide. 
     In some embodiments of the disclosed methods for selecting an amino acid motif that is glycosylated by an N-glycosyltransferase, the library of peptides comprises at least about 100, 500, 1000, 2000, 5000 or more peptides having a randomized sequence, where each peptide of the library has a different sequence. In some embodiments of the disclosed methods, the peptides comprise at least 6, 7, 8, or more amino acids and comprise at least a sequence X −2 -X −1 -N-X +1 -S/T-R-C wherein X is any amino acid. 
     In the disclosed methods for selecting an amino acid motif that is glycosylated by an N-glycosyltransferase, self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) is utilized to detect peptides that are glycosylated by the N-glycosyltransferase. Typically, the peptides are covalently immobilized on the self-assembled monolayers via a reaction between free maleimides present on the self-assembled monolayers and the C-terminal Cys of the peptides where the free maleimides and the C-terminal Cys react to form a covalent C—S bond. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 . Strategy for characterizing and designing glycosylation sites. Peptide acceptor and sugar donor preferences of N-glycosyltransferases from  A. pleuropneumoniae  (NGT),  Mannheimia haemolytica  and  Haemophilus ducreyi , along with two human polypeptide-N-acetylgalactosyltransferases, and the human O-GlcNAc transferase were characterized with 3,480 unique peptides and 13,903 unique reactions using SAMDI and enzyme produced by CFPS. Optimized sequences from NGT were used to design glycosylation sites on three heterologous proteins. Proteins were synthesized and glycosylated in vitro and in the cytoplasm of living  E. coli.    
         FIG. 2 : GlycoSCORES workflow and application to X −1  and X +1  position screening of NGT substrates. (a) Scheme of GlycoSCORES. NGT produced in CFPS was reacted with peptide acceptor and sugar donor in 384 well plates. Reaction products were applied directly to self-assembled maleimide functionalized alkanethiolate monolayers on gold islands. Peptides were captured to the monolayer by a terminal Cys residue via Michael Addition. MALDI-MS spectrum of this monolayer shows the addition of a single glucose residue (+162 Da) on a representative peptide. (b) NGT peptide acceptor preferences were determined at the X −1  and X +1  position relative to the modified Asn residue using the scheme in a. All NGT heat maps are arranged by mean glycosylation efficiency of each amino acid and the combination of amino acids at the other positions in descending order from left to right and top to bottom. NGT sequences showing high modification (blue boxes, &gt;66%) and low modification (grey boxes, &lt;3%) were chosen for subsequent screens towards an optimized GlycTag. Glycosylation reactions were performed with 50 μM peptide, 2.5 mM UDP-Glucose, and 0.2 μM CFPS NGT incubated at 30° C. for 3 h. Conversion efficiencies were determined by integration of mass spectra peaks at substrate and products masses and adjusted by measured relative ionization factor (RIFs) (data not shown). Numerical values of modification efficiencies from SAMDI-MS spectra were acquired from n=2 peptide immobilizations (data not shown). Control peptide maps also were generated using CFPS without NGT (data not shown). 
         FIG. 3 : Using GlycoSCORES to determine peptide selectivity of human ppGalNAcTs. (a) Specificity of ppGalNAcT1 produced in CFPS on peptide array of AX −1 TX +1 APRC designed from peptide AATPAP 38 . (b) The same peptide array with ppGalNAcT2 produced in CFPS. Reaction conditions: 100 μM peptide, 1 mM UDP-GalNAc, and 0.024 μM CFPS ppGalNAcT1 (a) or 0.04 μM CFPS ppGalNAcT2 (b) incubated at 37° C. for 1 h. Isoforms ppGalNAcT1 and ppGalNAcT2 share 40% sequence identity (Uniprot Clustal Omega Alignment 50 ) and showed major difference in peptide preferences. Peptide maps annotated with numerical values of the relative intensities of peptide substrates and glycosylated products and a negative control library generated using CFPS without ppGalNAcTs averaged from n=2 SAMDI-MS spectra from separate peptide immobilizations are shown in  FIG. 13 . 
         FIG. 4 : GlycoSCORES X +2 , X −2 , and X +3  position peptide specificity screening of NGT. (a) Highly modified peptide sequences from  FIG. 2 b    were tested with 19 amino acids in position X +2  relative to modified Asn. Only Thr and Ser showed efficient modification. (b) Sequences from  FIGS. 2 b  and 4 a    were tested with 19 amino acids in the X −2  position. Only the GNWT sequence showed efficient modification with all amino acids at the X −2  position. (c) Sequences from 4b were tested with 19 amino acids in the X +3  position. Sequences showing high (blue boxes, &gt;66%), medium (black boxes, 33-66%), and low (grey boxes, &lt;3%) modification efficiencies were chosen for subsequent screens towards an optimized GlycTag. Four sequences from (c) with varying levels of modification were selected for installation into proteins in  FIGS. 5 and 6 . Peptide sequences for which modification efficiencies could not be determined due to poor peptide solubility are shown as filled grey squares in the heat map. Reactions in (a-b) were completed with 0.2 μM NGT for 3 h. Reaction conditions were altered to 0.025 μM NGT for 1 h in (c) to avoid saturation. Peptide maps also were completed using 0.1 μM NGT for 1 h (data not shown). Numerical values of modification efficiencies from SAMDI-MS spectra were acquired from n=2 separate peptide immobilizations (data not shown). Control peptide arrays reacted with CFPS reactions without NGT also were generated (data not shown). 
         FIG. 5 : In vitro synthesis and glycosylation of Im7 with GlycoSCORES identified sequences. Im7 GlycTag variants and NGT were synthesized in CFPS then combined with UDP-glucose in an (IVG) reaction (workflow shown in  FIG. 19 ). (a) Representative LC-TOF spectra from analysis of n=3 IVG reactions generated by maximum entropy deconvolution of the Im7-6 variant containing the GlycoSCORES optimized GlycTag GGNWTT (SEQ ID NO:514) after Ni-NTA purification from IVG reactions with and without NGT. Representative deconvoluted spectra from all sequences and controls are shown in  FIG. 19 . Deconvolution mass errors as well as chromatogram peak retention times and quantification of relative peak area for all samples were generated and analyzed (data not shown). (b) Relative peak areas of Glc 1 /(Glc 0 +Glc 1 ) for Im7 variants containing different GlycTags. The results correlate with kinetics data measured by SAMDI for corresponding peptide sequences (inset c). Relative peak areas were calculated from extracted ion chromatograms of the 3 most abundant charge states based on theoretical average masses (see Methods). Mean and S.D. of n=3 IVG reactions are shown. * indicates significance by 2-tailed t-test with p-value of 0.016. Kinetic parameters of six peptide substrates (GGNWTTRC (SEQ ID NO:501), FANATTRC (SEQ ID NO:502), YANATSRC (SEQ ID NO:503), GANATARC (SEQ ID NO:504), DQNATFRC (SEQ ID NO:505), and NHETDRC (SEQ ID NO:506)) were analyzed (data not shown). Sequences positioned at Im7 internal loop are flanked by spacer sequences of the form ATT(X −2 X −1 NX +1 X +2 X +3 )AGG. Shading of column graph areas indicates increasing relative peak areas and therefore greater glycosylation efficiencies. 
         FIG. 6 : Site-directed cytoplasmic glycosylation of human Fc using GlycoSCORES optimized sequences. (a) Workflow for cytoplasmic glycosylation in living  E. coli  by coexpression of NGT and target protein GlycTag variants. (b) Relative peak areas of Glc 1 /(Glc 0 +Glc 1 ) for Fc containing naturally occurring (0), biological consensus (3), and GlycoSCORES optimized sequence (6) at Asn297 internal loop position with flanking sequences as in ATT(X −2 X −1 NX +1 X +2 X +3 )AGG. The GlycoSCORES identified GlycTag (GGNWTT (SEQ ID NO:514)) showed higher relative peak area, indicating greater glycosylation efficiency. Fc was treated with DTT for disulfide reduction before LC-TOF analysis. Relative peak areas calculated from extracted ion chromatograms of the 8 most abundant peaks based on theoretical average masses for Fc (see Methods). Mean and S.D. of n=3 cell cultures are shown. * and *** indicate significances by 2-tailed t-tests of p-values 0.017 and 0.00037, respectively. (c) Representative LC-TOF spectra from analysis of n=3 cell cultures generated by maximum entropy deconvolution are shown on the right (see Methods). Representative deconvolution spectra including N/Q and no NGT controls for are shown in  FIG. 20 . Deconvolution mass errors as well as chromatogram peak retention times and quantification of relative peak area for all samples were generated and analyzed (data not shown). A similar analysis of HMW1ct showed similar results with the GlycoSCORES optimized GlycTag providing more efficient glycosylation ( FIG. 22 ). Shading of bar areas indicates increasing relative peak areas and therefore greater glycosylation efficiencies. 
         FIG. 7 . Cell-free protein synthesis at 20° C. is &gt;95% complete after 20 h. Superfolder green fluorescent protein (sfGFP) expression over time at 20° C. Fluorescence of 15 μL CFPS reactions synthesizing sfGFP were incubated at 20° C., flash frozen on liquid nitrogen and measured on a 96-well plate fluorimeter. Average and S.D. of n=3 CFPS reactions are shown at each time-point. 
         FIG. 8 . CFPS provides high soluble expression titers of NGT and AGT. (A) Total and soluble yields of NGT and the α-1,6 glucose polymerase from  A. pleuropneumoniae  (AGT) from CFPS reactions were determined using  14 C-leucine incorporation. Results indicate high and consistent yields of both enzymes. Average and S.D. of n=3 CFPS reactions are shown. (B) An autoradiogram representative of n=2 experiments with similar results confirmed that the CFPS reactions primarily produced full-length product without large truncations. The autoradiogram was generated by a 48-h exposure of a 4-12% SDS-PAGE gel run in MOPS with NGT and AGT produced in CFPS with  14 C-leucine. Soluble samples were taken after centrifugation at 12,000×g for 15 min at 4° C. The same gel was Coomassie stained and aligned with autoradiogram image for molecular weight standard reference. 
         FIG. 9 . Purified NGT installs glucose onto short peptide sequences at asparagine residues. SAMDI mass spectra of peptide QNATFC (SEQ ID NO:510) before (A) and after (B) NGT treatment. A shift of +162 m/z was observed after NGT treatment. (C) Truncated versions of the previously studied GSDQNATF 9  peptide (SEQ ID NO:507) were modified with Cys at the N-terminal or C-terminal for SAMDI pull-down. The relative intensities of peptide substrates and glycosylated products, Glc 1 /(Glc 1 +Glc 0 ), observed in mass spectra with and without purified NGT are shown in the heat map. Peptides as small as CNAT (SEQ ID NO:508) and NATC (SEQ ID NO:509) could be modified by NGT and QNATFC (SEQ ID NO:510) showed the most efficient glucose modification. Reaction condition: 50 μM peptide, 2.5 mM UDP-Glc, and 0.5 μM purified NGT incubated at 30° C. for 3 h. Heat map shows the relative intensities calculated from SAMDI-MS spectra acquired from n=1 immobilization of each peptide. 
         FIG. 10 . NGT purified from living  E. coli  and synthesized by CFPS have similar peptide selectivity and specific activity. Percentage glucose modifications of X −1 NX +1 TRC peptide library was determined using NGT purified from living  E. coli  (A) and NGT produced by CFPS (B, the same data as  FIG. 2 b   ). The slight differences in conversion is near to expected variation (less than 0.10) between experiments with identical reaction conditions. Reaction conditions: 50 μM peptide, 2.5 mM UDP-Glc, and 0.2 μM NGT incubated for 3 h at 30° C. Heat maps show the average of n=2 SAMDI-MS spectra acquired from separate peptide immobilizations. 
         FIG. 11 . Sugar donor preferences of NGT. (A-C) Relative intensities of peptide substrates and glycosylated products observed in mass spectra of 6 peptides from X −1 NX +1 T library reacted with NGT and different sugar donors UDP-Glc, UDP-Gal, GDP-Man, UDP-GlcNAc, UDP-GalNAc and UDP-Xyl. The mean and S.D. of relative intensities of the 6 peptide modification values from heat maps are shown on the right. Only UDP-Glc, UDP-Gal and UDP-Xyl could be modified under these conditions, with UDP-Glc strongly preferred. All reactions were conducted with 50 μM peptide and 1 mM sugar donor, reacted with 0.1 μM purified NGT incubated at 30° C. for 1 h (A), 0.1 μM purified NGT incubated at 30° C. for 4 h (B), or 0.2 μM purified NGT incubated at 30° C. for 21 h (C). (D-F) Relative intensities of peptide substrates and glycosylated products observed in mass spectra for the X −1 NX +1 TRC peptide library reacted with UDP-Glc (D), UDP-Gal (E) and UDP-Xyl (F). Peptide preferences are weakly dependent on sugar donors UDP-Glc, UDP-Gal, and UDP-Xyl. For example, NGT is less tolerant of Trp at the X −1  position when transferring xylose compared to glucose or galactose. All reactions were conducted with 50 μM peptide and 1 mM sugar donor and reacted with 0.1 μM purified NGT incubated at 30° C. for 4 h (D), 10 μM purified NGT incubated at 30° C. for 21 h (E), or 4 μM purified NGT incubated at 30° C. for 21 h (F). To ensure that endogenous activated sugar donors or metabolic processes in CFPS did not provide false positives of galactose transfer, sugar donor experiments were completed with purified NGT. Relative intensities were calculated using I(P)/(I(S)+I(P)) where I(P) is the MS intensity of the glycosylated product and I(S) is the MS intensity of the aglycosylated substrate. Heat maps show the average of n=2 SAMDI-MS spectra acquired from separate peptide immobilizations. 
         FIG. 12 . Percent identity matrix and alignment of NGT homologs used in this study. (A) Percent identity matrix of NGT homologs characterized in this study, NGT (Uniprot: NGT_ACTP2), MhNGT (Uniprot: A0A0B5BRN9_MANHA), and HdNGT (Uniprot: Q7VKK3_HAEDU). (B) CLUSTAL OMEGA alignment of NGT homologs showing levels of conservation across enzymes. 
         FIG. 13 . Peptide specificity maps of ppGalNAcTs annotated with numerical values and peptide control library. (A) Peptide specificity map of CFPS GalNAcT1 shown in  FIG. 3 a    annotated with numerical values of the relative intensities peptide substrates and glycosylated products. (B) The same peptide array and annotations with CFPS ppGalNAcT2 shown in  FIG. 3 b   . (C) Negative control library incubated with CFPS sfGFP showed no GalNAc modification. Reaction conditions: 100 μM peptide, 1 mM UDP-GalNAc, and 0.024 mM CFPS ppGalNAcT1 (A), 0.04 mM CFPS ppGalNAcT2 (B) or CFPS sfGFP (C) incubated at 37° C. for 1 h. Isoforms ppGalNAcT1 and ppGalNAcT2 share 40% (Uniprot Clustal Omega Alignment 15 ) sequence identity and showed major differences in peptide preferences. Heat maps show the average of n=2 SAMDI-MS spectra from separate peptide immobilizations. 
         FIG. 14 . GlycoSCORES characterization of human OGT. Peptide specificity map of human OGT produced in CFPS. We analyzed 19-amino acid substitutions for each position of a previously proposed OGT targeting sequence of PPVSR and also investigated the specificity at the X −4  and X +2 . This screen shows successful production of active human OGT in CFPS and that GlycoSCORES can be used to probe peptide specificity of hOGT in high throughput. This is the first saturated substitution of this sequence, and specificity results were generally consistent with previous analysis of human glycosylation sites and peptide screens 11, 16, 17 , such as the preference for V in the X −3  position and A in the X +2  position. When we extended the sequence to include an X −4  amino acid, we observed an overall increase in modification and a strong preference for aromatic residues. This result indicates that the presence and identity of the X −4  position is important for OGT activity. Reaction conditions: 50 μM peptide, 2.5 mM UDP-GlcNAc, and 0.062 μM CFPS hOGT. The heat map shows the average of n=2 SAMDI-MS spectra from separate peptide immobilizations. 
         FIG. 15 . Amino acids at X +2  position weakly influence amino acid selectivity at X −1  and X +1  positions. (A) Percentage glucose modification map of peptide library X −1 NX +1 TRC using CFPS NGT, the same as  FIG. 2 b   . (B) Percentage glucose modification map of peptide library X −1 NX +1 SRC using CFPS NGT. Modification patterns are nearly identical, but T is preferred over S in the X +2  position. Reaction condition: 50 μM peptide, 2.5 mM UDP-Glc, and 0.2 μM CFPS NGT incubated at 30° C. for 3 h. Heat maps show the average of n=2 SAMDI-MS spectra acquired from separate peptide immobilizations. 
         FIG. 16 . NGT only efficiently modifies peptides with Asn at the X 0  position. A 19-peptide library with naturally occurring amino acids substituted into YANATTRC (SEQ ID NO:511) at the X 0  position was synthesized and evaluated for NGT activity at three reaction conditions spanning the range of conditions relevant to GlycoSCORES analysis. Only Asn at X 0  position showed detectable modification. All reactions were conducted with 50 μM peptide and 2.5 mM UDP-Glc, reacted with 0.025 μM NGT synthesized in CFPS incubated at 30° C. for 1 h (1), 0.1 μM NGT synthesized in CFPS incubated at 30° C. for 1 h (1), or 0.5 μM NGT synthesized in CFPS incubated at 30° C. for 21 h (3). Heat maps show the average of n=2 SAMDI-MS spectra acquired from separate peptide immobilizations. 
         FIG. 17 . GlycoSCORES optimized GlycTag peptides are more efficiently modified than previously studied and naturally occurring glycosylation sequences. (A) Comparison of glucose modification of GlycoSCORES identified sequences with high (blue boxes), medium (black boxes), and low (grey boxes) modification efficiency to peptide sequences found to have N-glucose modifications in previous NGT studies 9, 18-20  (B) Modification efficiencies measured by GlycoSCORES of naturally occurring N-glycosylation sequences used in this study from  H. influenzae  HMW1 protein (NINATS (SEQ ID NO:512)) and  H. sapiens  IgG (QYNSTY (SEQ ID NO:513)). Modification efficiencies for GlycoSCORES optimized GlycTag (GGNWTT, GlycTag ID: 6 (SEQ ID NO:514)) and a biological consensus sequence (GANATA, GlycTag ID: 3 (SEQ ID NO:515)) found in a previous study 18  are also shown on the left. All reactions were conducted with 50 μM peptide and 2.5 mM UDP-Glc, reacted with 0.025 μM NGT synthesized in CFPS incubated at 30° C. for 1 h (1) or 0.1 μM NGT synthesized in CFPS incubated at 30° C. for 1 h (2). Heat maps show the average of n=2 SAMDI-MS spectra acquired from separate peptide immobilizations. 
         FIG. 18 . In vitro recapitulation of  A. pleuropneumoniae  multi-enzyme glycosylation system by synthesis and glycosylation of HMW1ct-WT. (A) Scheme for IVG of HMW1ct-WT by NGT and AGT. HMW1ct-WT, NGT, and AGT were synthesized in separate CFPS reactions for 20 h and combined with UDP-Glc or glucose. IVG reactions were performed with 5 μM HMW1ct-WT, 1 μM NGT, 2 μM AGT, and 2.5 mM UDP-Glc or 200 mM glucose for 16 h. The concentration of soluble HMW1ct-WT in completed CFPS reactions incubated for 20 h at 20° C. was determined by  14 C-leucine incorporation to be 701±32 μg/mL. (B) Reaction scheme for glucose modification of HMW1ct-WT with NGT and AGT. (C) On an α-His western blot representative of n=2 experiments with similar results, we observed small gel mobility shifts in lanes with NGT (indicating single glucose modification of up to 12 available N-X-S/T glycosylation sites) and high molecular weight bands in lanes with NGT and AGT (indicating polyglucose modifications). We found that glycosylation was achieved with both UDP-Glc substrate as well simple glucose, albeit to a lower extent. This is likely due to conversion of glucose to UDP-Glc by endogenous  E. coli  enzymes present in the crude  E. coli  lysate. Poly-α-glucose modifications were confirmed by corresponding signals on ConA blot which specifically binds to terminal α-linked glucose residues. * denotes control lane loaded with CFPS that synthesized sfGFP, ** denotes control lane loaded with CFPS reaction that synthesized sfGFP plus NGT and AGT synthesized in CFPS. 
         FIG. 19 . Scheme and deconvoluted MS spectra from LC-MS analysis of Im7 variants synthesized and glycosylated in vitro. (A) Workflow for rapid synthesis and glycosylation of protein variants. Protein variants and NGT are synthesized in CFPS then assembled into IVG reactions with 5 μM of the indicated Im7 variant produced in CFPS, 0.1 μM NGT produced in CFPS (unless concentration difference is noted; see F), and 2.5 mM UDP-Glc and incubated for 2.5 h at 30° C. Im7 was purified by Ni-NTA and injected into LC-TOF. (B) Representative deconvoluted spectra of proteins from analysis of n=3 IVG reaction products with indicated GlycTag sequences identified by GlycoSCORES analysis. All sequences were flanked by ATT-(X −2 X −1 NX +1 X +2 X +3 )-AGG. Trends of protein glycosylation match observed modification efficiencies and reaction kinetics constants determined by GlycoSCORES at the peptide level. (C) Deconvoluted spectrum of Im7 variant without flanking sequences shows no detectable glycosylation, indicating flanking sequences are required for efficient modification under these conditions. (D) Deconvoluted spectrum of N/Q substituted Im7 variant shows no detectable glycosylation, confirming that the redesigned target site was the only site efficiently modified by NGT. (E) Deconvoluted spectrum of Im7 purified from an IVG reaction containing no NGT shows no detectable glycosylation, indicating that the single glucose modification is performed by NGT. (F) Deconvoluted spectrum of Im7 bearing the GlycoSCORES optimized sequence GGNWTT (SEQ ID NO:514) purified from an IVG reaction containing 4 μM NGT with complete glycosylation. (C-F) spectra are representative of n=2 IVGs with similar results. All spectra processed by Agilent Mass Hunter Max Entropy Deconvolution of 700-2000 m/z range into 10,000-15,000 u mass range. 
         FIG. 20 . Deconvoluted mass spectra from LC-TOF analysis of Fc variants synthesized and glycosylated in the cytoplasm of living  E. coli . Sequence variants of the Fc target protein were coexpressed with NGT in BL21(DE3)  E. coli , purified by Ni-NTA, reduced by DTT and injected into LC-TOF. The Fc target protein was induced with 400 μM IPTG for 2 h (unless noted; see E), followed by a 30 min induction of NGT with 0.2% arabinose. (A) Representative deconvoluted spectra from n=3 expression cultures of Fc containing the indicated naturally occurring (0), biological consensus 18  (3), and optimized GlycTag (6) sequences are shown. All glycosylation sequences are flanked by flexible linker sequences of the form ATT-(X −2 X −1 NX +1 X +2 X +3 )-AGG. Protein modification trends match those predicted by GlycoSCORES of peptides and show that the protein containing the optimized GlycTag sequence is the most efficiently modified (spectra also shown in  FIG. 6 ). (B) Deconvoluted spectrum of Fc-0s variant without flanking sequences showed no modification, indicating that flanking sequences were required for efficient modification under these conditions. (C) Deconvoluted spectrum of an Fc variant with N/Q substitution to remove the glycosylation site of Fc-0 showed no detectable glycosylation, indicating that only the redesigned site was efficiently modified. (D) Deconvoluted spectrum of Fc-6 expressed in BL21(DE3)  E. coli  with no NGT plasmid present showed no detectable glycosylation, confirming that NGT was responsible for the glucose modification. (E) Deconvoluted spectrum of the Fc variant engineered with the optimized GlycTag co-expressed with NGT for 4 h showed complete glycosylation, indicating that NGT can be used to create homogeneously modified Fc in the cytoplasm of living  E. coli . (B-E) spectra are representative of analysis of n=2 expression cultures with similar results. All spectra processed by Agilent Mass Hunter Max Entropy Deconvolution of 700-2000 m/z range into mass range of 25,000-30,000 u. 
         FIG. 21 . In vitro synthesis and glycosylation of Fc. Sequence variants of the Fc target protein were expressed in CFPS for 20 h and glycosylated in vitro using NGT expressed in CFPS. Results show that Fc engineered with optimized GlycoSCORES GlycTag can be efficiently glycosylated in vitro. Unless otherwise noted, each IVG reaction contained 5 μM Fc variant, 4 μM NGT, and 2.5 mM UDP-Glc and was incubated for 2.5 h at 30° C. then purified by Ni-NTA magnetic beads and injected into Q-TOF. Fc and NGT concentration in IVG quantified by  14 C-leucine incorporation. (A) Deconvoluted spectra of Fc-0 variant bearing the naturally occurring glycosylation sequence showed no detectable glycosylation. (B) Deconvoluted spectra of Fc-6 variant bearing optimized GlycTag showing efficient glycosylation. (C) Deconvoluted spectra of Fc-6 variant bearing optimized GlycTag from 12 h IVG reaction showing nearly homogeneous glycosylation. (D) Deconvoluted spectra of Fc-6 variant bearing optimized GlycTag in IVG reaction condition without NGT showing that modification is due to NGT. (E) Deconvoluted spectra of Fc-N/Q variant showing no glycosylation at alternative sites. All spectra representative of n=2 IVGs. Spectra were processed by Bruker Compass Max Entropy Deconvolution of 700-3000 m/z range into mass range of 20,000-30,000 u. 
         FIG. 22 . Deconvoluted mass spectra from LC-MS analysis of HMW1ct variants synthesized and glycosylated in the cytoplasm of living  E. coli . The HMW1ct glycosylation site at Asn1366 was redesigned to the biological consensus sequence (GANATA) 18  (SEQ ID NO:515) or the GlycoSCORES optimized GlycTag (GGNWTT (SEQ ID NO:514)). All other Asn residues within N-X-S/T motifs were mutated to Gln, generating a variant with only one site available for efficient glycosylation by NGT. Variants of the HMW1ct target protein were induced for 1 h followed by a 2 h induction of NGT in BL21(DE3)  E. coli  then purified by Ni-NTA and injected into LC-TOF. (A) Representative deconvoluted spectra of n=3 expression cultures HMW1ct containing the indicated naturally occurring (0), biological consensus 18  (3), and optimized GlycTag (6) sequences are shown. Protein modification trends match those predicted by GlycoSCORES of peptides and show that the protein containing the optimized GlycTag sequence is the most efficiently modified. (B) Deconvoluted spectra of HMW1ct-0 variant with N/Q substitution to remove glycosylation site showed no detectable glycosylation, indicating that only the redesigned site was efficiently modified. (C) Deconvoluted spectrum of HMW1ct in BL21(DE3) strain with no NGT plasmid present shows no detectable glycosylation. Sequence variants of the HMW1ct target protein were induced for 1 h followed by a 2 h induction of NGT. (A-C) spectra representative of analysis of n=3 cell cultures. (B-C) spectra are representative of n=2 expression cultures. All spectra processed by Agilent Mass Hunter Max Entropy Deconvolution of 700-2000 m/z range into mass range of 32,500-37,500 u. (D) Relative peak areas of Glc 1 /(Glc 0 +Glc 1 ) for HMW1ct containing naturally occurring (0), biological consensus 18  (3), and GlycoSCORES optimized sequence (6) at Asn1366 position. Relative peak areas calculated from extracted ion chromatograms of the 10 most abundant peaks based on theoretical average masses (see Methods). Average and S.D. of triplicate (n=3) cell cultures are shown. ** and *** indicate significances by 2-tailed t-tests of p-values 0.0035 and 0.00037, respectively. 
         FIG. 23 . Site-specific control of glycosylation by rapid enzyme characterization and sequential addition of enzymes. Four NGT homologs were selected from a phylogenetic screen of putative NGT enzymes and characterized using GlycoSCORES to find differences in peptide specificity. Peptide sequences showing conditional orthogonality which would enable site-specific glycosylation when applied in the correct order, were discovered by further GlycoSCORES optimization. Optimized GlycTags were incorporated into a single glycoprotein and NGTs were added sequentially to site-specifically control glycosylation at up to four glycosylation sites within one protein. 
         FIG. 24 . Phylogenetic tree of 41 putative NGTs from CAZY database which were screened for N-glycosyltransferase activity by expression and CFPS and analysis by SAMDI-MS. 
         FIG. 25 . Optimized GlycTag sequences show conditional orthogonality at peptide level and enable differential targeting of glycosylation sites within protein. (a) Conditional orthogonality of optimized 6-mer GlycTags. Selected GlycTags were screened for HiNGT, EcNGT, ApNGT and ApNGT Q469A  modification by SAMDI in triplicate experiments. Heat map shows conditional orthogonality. Experimental conditions: 0.2 μM purified HiNGT or 0.67 μM purified EcNGT, 30° C. for 21 h; 0.45 μM purified ApNGT or 0.1 μM purified ApNGT Q469A , 30° C. for 3 h. (b) Optimized 6-mer GlycTags were inserted into the N-terminus, C-terminus, and two exposed loops of the glycosylation model protein Im7, with flanking sequences of RATT (SEQ ID NO:516)-GlycTag-AGGR (SEQ ID NO:517) to facilitate trypsinization and quantitative LC-MS analysis. (c) Differential targeting of four optimized GlycTags within a single Im7 target protein. Im7 bearing the four optimized GlycTags was reacted with 2.5 mM UDP-Glucose and various concentrations of each purified NGT for 4 h. After the modification, Im7 was purified using Ni-NTA functionalized magnetic beads, treated with trypsin and analyzed by LC-qTOF. (d) Heatmap showing conditional orthogonality of each NGT for 6-mer GlycTags within Im7 under optimized conditions. Experimental conditions: 0.3 μM purified HiNGT, 0.3 μM purified EcNGT, 0.04 μM purified ApNGT or 0.04 μM purified ApNGT Q469A , 30° C. for 4 h. All are experimental triplicate. Similar differential modification patterns by different NGTs were observed for peptide sequences and GlycTags within an engineered Im7 acceptor protein. 
         FIG. 26 . Site-specific control of glycosylation at four distinct GlycTag sequences within one target protein. (a) A workflow for differential control of glycosylation at four sites within Im7 by sequential addition of NGT enzymes. (b) The intact Im7 MS spectra and bar graph percentage of nGlc-Im7 after each step; triplicate. (c) A bar graph of modification of each of the four GlycTags after each step and trypsinization of the target protein bearing four distinct GlycTags; triplicate. 
     
    
    
     DETAILED DESCRIPTION 
     Definitions and Terminology 
     The disclosed components, systems, and methods for glycoprotein and recombinant glycoprotein protein synthesis may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting. 
     As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a oligosaccharide” or “an N-glycosyltransferase” should be interpreted to mean “one or more oligosaccharides” and “one or more N-glycosyltransferase,” respectively, unless the context clearly dictates otherwise. As used herein, the term “plurality” means “two or more.” 
     As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term. 
     As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. 
     The phrase “such as” should be interpreted as “for example, including.” Moreover the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. 
     Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.” 
     All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth. 
     The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.” 
     As used herein, the terms “bind,” “binding,” “interact,” “interacting,” “occupy” and “occupying” refer to covalent interactions, noncovalent interactions and steric interactions. A covalent interaction is a chemical linkage between two atoms or radicals formed by the sharing of a pair of electrons (a single bond), two pairs of electrons (a double bond) or three pairs of electrons (a triple bond). Covalent interactions are also known in the art as electron pair interactions or electron pair bonds. Noncovalent interactions include, but are not limited to, van der Waals interactions, hydrogen bonds, weak chemical bonds (via short-range noncovalent forces), hydrophobic interactions, ionic bonds and the like. A review of noncovalent interactions can be found in Alberts et al., in Molecular Biology of the Cell, 3d edition, Garland Publishing, 1994. Steric interactions are generally understood to include those where the structure of the compound is such that it is capable of occupying a site by virtue of its three dimensional structure, as opposed to any attractive forces between the compound and the site. 
     Polynucleotides and Synthesis Methods 
     The terms “nucleic acid” and “oligonucleotide,” as used herein, refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs. 
     Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979 , Meth. Enzymol.  68:90-99; the phosphodiester method of Brown et al., 1979 , Meth. Enzymol.  68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981 , Tetrahedron Letters  22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each incorporated herein by reference. A review of synthesis methods of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild, 1990 , Bioconjugate Chemistry  1(3): 165-187, incorporated herein by reference. 
     The term “amplification reaction” refers to any chemical reaction, including an enzymatic reaction, which results in increased copies of a template nucleic acid sequence or results in transcription of a template nucleic acid. Amplification reactions include reverse transcription, the polymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), and the ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary “amplification reactions conditions” or “amplification conditions” typically comprise either two or three step cycles. Two-step cycles have a high temperature denaturation step followed by a hybridization/elongation (or ligation) step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step. 
     The terms “target,” “target sequence”, “target region”, and “target nucleic acid,” as used herein, are synonymous and refer to a region or sequence of a nucleic acid which is to be amplified, sequenced, or detected. 
     The term “hybridization,” as used herein, refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzy et al., 2008 , Biochemistry,  47: 5336-5353, which are incorporated herein by reference). 
     The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. 
     A primer is preferably a single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 150 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein. 
     Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis. For example, primers may contain an additional nucleic acid sequence at the 5′ end which does not hybridize to the target nucleic acid, but which facilitates cloning or detection of the amplified product, or which enables transcription of RNA (for example, by inclusion of a promoter) or translation of protein (for example, by inclusion of a 5′-UTR, such as an Internal Ribosome Entry Site (IRES) or a 3′-UTR element, such as a poly(A) n  sequence, where  n  is in the range from about 20 to about 200). The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region. 
     As used herein, a primer is “specific,” for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily to the target nucleic acid. Typically, a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample. One of skill in the art will recognize that various factors, such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be needed in many cases. Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence. Thus, the use of target-specific primers under suitably stringent amplification conditions enables the selective amplification of those target sequences that contain the target primer binding sites. 
     As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example,  Pyrococcus furiosus  (Pfu) DNA polymerase,  E. coli  DNA polymerase I, T7 DNA polymerase and  Thermus aquaticus  (Taq) DNA polymerase, among others. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and  E. coli  RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art. 
     The term “promoter” refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence. 
     As used herein, the term “sequence defined biopolymer” refers to a biopolymer having a specific primary sequence. A sequence defined biopolymer can be equivalent to a genetically-encoded defined biopolymer in cases where a gene encodes the biopolymer having a specific primary sequence. 
     The polynucleotide sequences contemplated herein may be present in expression vectors. For example, the vectors may comprise: (a) a polynucleotide encoding an ORF of a protein; (b) a polynucleotide that expresses an RNA that directs RNA-mediated binding, nicking, and/or cleaving of a target DNA sequence; and both (a) and (b). The polynucleotide present in the vector may be operably linked to a prokaryotic or eukaryotic promoter. “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. Vectors contemplated herein may comprise a heterologous promoter (e.g., a eukaryotic or prokaryotic promoter) operably linked to a polynucleotide that encodes a protein. A “heterologous promoter” refers to a promoter that is not the native or endogenous promoter for the protein or RNA that is being expressed. Vectors as disclosed herein may include plasmid vectors. 
     As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. 
     As used herein, “expression template” refers to a nucleic acid that serves as substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein). Expression templates include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use a nucleic acid for an expression template include genomic DNA, cDNA and RNA that can be converted into cDNA. Genomic DNA, cDNA and RNA can be from any biological source, such as a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecal sample, a urine sample, a scraping, among others. The genomic DNA, cDNA and RNA can be from host cell or virus origins and from any species, including extant and extinct organisms. As used herein, “expression template” and “transcription template” have the same meaning and are used interchangeably. 
     In certain exemplary embodiments, vectors such as, for example, expression vectors, containing a nucleic acid encoding one or more rRNAs or reporter polypeptides and/or proteins described herein are provided. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably. However, the disclosed methods and compositions are intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. 
     In certain exemplary embodiments, the recombinant expression vectors comprise a nucleic acid sequence (e.g., a nucleic acid sequence encoding one or more rRNAs or reporter polypeptides and/or proteins described herein) in a form suitable for expression of the nucleic acid sequence in one or more of the methods described herein, which means that the recombinant expression vectors include one or more regulatory sequences which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence encoding one or more rRNAs or reporter polypeptides and/or proteins described herein is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription and/or translation system). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). 
     Oligonucleotides and polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, S 2 T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone. 
     The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand). 
     Regarding polynucleotide sequences, the terms “percent identity” and “% identity” refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above). 
     Regarding polynucleotide sequences, percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured. 
     Regarding polynucleotide sequences, “variant,” “mutant,” or “derivative” may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information&#39;s website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair of nucleic acids may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. 
     Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code where multiple codons may encode for a single amino acid. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. For example, polynucleotide sequences as contemplated herein may encode a protein and may be codon-optimized for expression in a particular host. In the art, codon usage frequency tables have been prepared for a number of host organisms including humans, mouse, rat, pig,  E. coli , plants, and other host cells. 
     A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. 
     The nucleic acids disclosed herein may be “substantially isolated or purified.” The term “substantially isolated or purified” refers to a nucleic acid that is removed from its natural environment, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it is naturally associated. 
     Peptides, Polypeptides, Proteins, and Synthesis Methods 
     As used herein, the terms “peptide,” “polypeptide,” and “protein,” refer to molecules comprising a chain a polymer of amino acid residues joined by amide linkages. The term “amino acid residue,” includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include nonstandard or unnatural amino acids. The term “amino acid residue” may include alpha-, beta-, gamma-, and delta-amino acids. 
     In some embodiments, the term “amino acid residue” may include nonstandard or unnatural amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, β-alanine, β-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. The term “amino acid residue” may include L isomers or D isomers of any of the aforementioned amino acids. 
     Other examples of nonstandard or unnatural amino acids include, but are not limited, to a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, an O-methyl-L-tyrosine, a p-propargyloxyphenylalanine, a p-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcpβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a methionine amino acid; an unnatural analogue of a leucine amino acid; an unnatural analogue of a isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, 33ufa33hor, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid; an α,α disubstituted amino acid; a β-amino acid; a γ-amino acid, a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan. 
     As used herein, a “peptide” is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett &amp; Grisham, Biochemistry, 2 nd  edition, 1999, Brooks/Cole, 110). In some embodiments, a peptide as contemplated herein may include no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. A polypeptide, also referred to as a protein, is typically of length ≥100 amino acids (Garrett &amp; Grisham, Biochemistry, 2 nd  edition, 1999, Brooks/Cole, 110). A polypeptide, as contemplated herein, may comprise, but is not limited to, 100, 101, 102, 103, 104, 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues. 
     A peptide as contemplated herein may be further modified to include non-amino acid moieties. Modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine). 
     The modified amino acid sequences that are disclosed herein may include a deletion in one or more amino acids. As utilized herein, a “deletion” means the removal of one or more amino acids relative to the native amino acid sequence. The modified amino acid sequences that are disclosed herein may include an insertion of one or more amino acids. As utilized herein, an “insertion” means the addition of one or more amino acids to a native amino acid sequence. The modified amino acid sequences that are disclosed herein may include a substitution of one or more amino acids. As utilized herein, a “substitution” means replacement of an amino acid of a native amino acid sequence with an amino acid that is not native to the amino acid sequence. For example, the modified amino sequences disclosed herein may include one or more deletions, insertions, and/or substitutions in order modified the native amino acid sequence of a target protein to include one or more heterologous amino acid motifs that are glycosylated by an N-glycosyltransferase. 
     Regarding proteins, a “deletion” refers to a change in the amino acid sequence that results in the absence of one or more amino acid residues. A deletion may remove at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, or more amino acids residues. A deletion may include an internal deletion and/or a terminal deletion (e.g., an N-terminal truncation, a C-terminal truncation or both of a reference polypeptide). A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a deletion relative to the reference polypeptide sequence. 
     Regarding proteins, “fragment” is a portion of an amino acid sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous amino acid residues of a reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a reference polypeptide. Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full-length polypeptide. A fragment may include an N-terminal truncation, a C-terminal truncation, or both truncations relative to the full-length protein. A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a fragment of the reference polypeptide sequence. 
     Regarding proteins, the words “insertion” and “addition” refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more amino acid residues. A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include an insertion or addition relative to the reference polypeptide sequence. A variant of a protein may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions. 
     Regarding proteins, the phrases “percent identity” and “% identity,” refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases. 
     Regarding proteins, percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured. 
     Regarding proteins, the amino acid sequences of variants, mutants, or derivatives as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative protein may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. The following table provides a list of exemplary conservative amino acid substitutions which are contemplated herein: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Original 
                 Conservative 
               
               
                   
                 Residue 
                 Substitution 
               
               
                   
                   
               
             
            
               
                   
                 Ala 
                 Gly, Ser 
               
               
                   
                 Arg 
                 His, Lys 
               
               
                   
                 Asn 
                 Asp, Gln, His 
               
               
                   
                 Asp 
                 Asn, Glu 
               
               
                   
                 Cys 
                 Ala, Ser 
               
               
                   
                 Gln 
                 Asn, Glu, His 
               
               
                   
                 Glu 
                 Asp, Gln, His 
               
               
                   
                 Gly 
                 Ala 
               
               
                   
                 His 
                 Asn, Arg, Gln, Glu 
               
               
                   
                 Ile 
                 Leu, Val 
               
               
                   
                 Leu 
                 Ile, Val 
               
               
                   
                 Lys 
                 Arg, Gln, Glu 
               
               
                   
                 Met 
                 Leu, Ile 
               
               
                   
                 Phe 
                 His, Met, Lett, Trp, Tyr 
               
               
                   
                 Ser 
                 Cys, Thr 
               
               
                   
                 Thr 
                 Ser, Val 
               
               
                   
                 Trp 
                 Phe, Tyr 
               
               
                   
                 Tyr 
                 His, Phe, Trp 
               
               
                   
                 Val 
                 Ile, Leu, Thr 
               
               
                   
                   
               
            
           
         
       
     
     Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. Non-conservative amino acids typically disrupt (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. 
     The disclosed proteins, mutants, variants, or described herein may have one or more functional or biological activities exhibited by a reference polypeptide (e.g., one or more functional or biological activities exhibited by wild-type protein). 
     The disclosed proteins may be substantially isolated or purified. The term “substantially isolated or purified” refers to proteins that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated. 
     Cell-Free Protein Synthesis (CFPS) 
     The components, systems, and methods disclosed herein may be applied to cell-free protein synthesis methods as known in the art. See, for example, U.S. Pat. Nos. 5,478,730; 5,556,769; 5,665,563; 6,168,931; 6,548,276; 6,869,774; 6,994,986; 7,118,883; 7,186,525; 7,189,528; 7,235,382; 7,338,789; 7,387,884; 7,399,610; 7,776,535; 7,817,794; 8,703,471; 8,298,759; 8,715,958; 8,734,856; 8,999,668; and 9,005,920. See also U.S. Published Application Nos. 2018/0016614, 2018/0016612, 2016/0060301, 2015-0259757, 2014/0349353, 2014-0295492, 2014-0255987, 2014-0045267, 2012-0171720, 2008-0138857, 2007-0154983, 2005-0054044, and 2004-0209321. See also U.S Published Application Nos. 2005-0170452; 2006-0211085; 2006-0234345; 2006-0252672; 2006-0257399; 2006-0286637; 2007-0026485; 2007-0178551. See also Published PCT International Application Nos. 2003/056914; 2004/013151; 2004/035605; 2006/102652; 2006/119987; and 2007/120932. See also Jewett, M. C., Hong, S. H., Kwon, Y. C., Martin, R. W., and Des Soye, B. J. 2014, “Methods for improved in vitro protein synthesis with proteins containing non standard amino acids,” U.S. Patent Application Ser. No. 62/044,221; Jewett, M. C., Hodgman, C. E., and Gan, R. 2013, “Methods for yeast cell-free protein synthesis,” U.S. Patent Application Ser. No. 61/792,290; Jewett, M. C., J. A. Schoborg, and C. E. Hodgman. 2014, “Substrate Replenishment and Byproduct Removal Improve Yeast Cell-Free Protein Synthesis,” U.S. Patent Application Ser. No. 61/953,275; and Jewett, M. C., Anderson, M. J., Stark, J. C., Hodgman, C. E. 2015, “Methods for activating natural energy metabolism for improved yeast cell-free protein synthesis,” U.S. Patent Application Ser. No. 62/098,578. See also Guarino, C., &amp; DeLisa, M. P. (2012). A prokaryote-based cell-free translation system that efficiently synthesizes glycoproteins. Glycobiology, 22(5), 596-601. The contents of all of these references are incorporated in the present application by reference in their entireties. 
     In some embodiments, a “CFPS reaction mixture” typically may contain a crude or partially-purified cell extract, an RNA translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the RNA translation template. In some aspects, the CFPS reaction mixture can include exogenous RNA translation template. In other aspects, the CFPS reaction mixture can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA-dependent RNA polymerase. In these other aspects, the CFPS reaction mixture can also include a DNA-dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame. In these other aspects, additional NTP&#39;s and divalent cation cofactor can be included in the CFPS reaction mixture. A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of ordinary skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of ordinary skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components of the invention. 
     The disclosed cell-free protein synthesis systems may utilize components that are crude and/or that are at least partially isolated and/or purified. As used herein, the term “crude” may mean components obtained by disrupting and lysing cells and, at best, minimally purifying the crude components from the disrupted and lysed cells, for example by centrifuging the disrupted and lysed cells and collecting the crude components from the supernatant and/or pellet after centrifugation. The term “isolated or purified” refers to components that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated. 
     As used herein, “translation template” for a polypeptide refers to an RNA product of transcription from an expression template that can be used by ribosomes to synthesize polypeptides or proteins. 
     The term “reaction mixture,” as used herein, refers to a solution containing reagents necessary to carry out a given reaction. A reaction mixture is referred to as complete if it contains all reagents necessary to perform the reaction. Components for a reaction mixture may be stored separately in separate container, each containing one or more of the total components. Components may be packaged separately for commercialization and useful commercial kits may contain one or more of the reaction components for a reaction mixture. 
     A reaction mixture may include an expression template, a translation template, or both an expression template and a translation template. The expression template serves as a substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein). The translation template is an RNA product that can be used by ribosomes to synthesize the sequence defined biopolymer. In certain embodiments the platform comprises both the expression template and the translation template. In certain specific embodiments, the reaction mixture may comprise a coupled transcription/translation (“Tx/Tl”) system where synthesis of translation template and a sequence defined biopolymer from the same cellular extract. 
     The reaction mixture may comprise one or more polymerases capable of generating a translation template from an expression template. The polymerase may be supplied exogenously or may be supplied from the organism used to prepare the extract. In certain specific embodiments, the polymerase is expressed from a plasmid present in the organism used to prepare the extract and/or an integration site in the genome of the organism used to prepare the extract. 
     Altering the physicochemical environment of the CFPS reaction to better mimic the cytoplasm can improve protein synthesis activity. The following parameters can be considered alone or in combination with one or more other components to improve robust CFPS reaction platforms based upon crude cellular extracts (for examples, S12, S30 and S60 extracts). 
     The temperature may be any temperature suitable for CFPS. Temperature may be in the general range from about 10° C. to about 40° C., including intermediate specific ranges within this general range, include from about 15° C. to about 35° C., from about 15° C. to about 30° C., from about 15° C. to about 25° C. In certain aspects, the reaction temperature can be about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C. 
     The reaction mixture may include any organic anion suitable for CFPS. In certain aspects, the organic anions can be glutamate, acetate, among others. In certain aspects, the concentration for the organic anions is independently in the general range from about 0 mM to about 200 mM, including intermediate specific values within this general range, such as about 0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM and about 200 mM, among others. 
     The reaction mixture may include any halide anion suitable for CFPS. In certain aspects the halide anion can be chloride, bromide, iodide, among others. A preferred halide anion is chloride. Generally, the concentration of halide anions, if present in the reaction, is within the general range from about 0 mM to about 200 mM, including intermediate specific values within this general range, such as those disclosed for organic anions generally herein. 
     The reaction mixture may include any organic cation suitable for CFPS. In certain aspects, the organic cation can be a polyamine, such as spermidine or putrescine, among others. Preferably polyamines are present in the CFPS reaction. In certain aspects, the concentration of organic cations in the reaction can be in the general about 0 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 2 mM. In certain aspects, more than one organic cation can be present. 
     The reaction mixture may include any inorganic cation suitable for CFPS. For example, suitable inorganic cations can include monovalent cations, such as sodium, potassium, lithium, among others; and divalent cations, such as magnesium, calcium, manganese, among others. In certain aspects, the inorganic cation is magnesium. In such aspects, the magnesium concentration can be within the general range from about 1 mM to about 50 mM, including intermediate specific values within this general range, such as about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, among others. In preferred aspects, the concentration of inorganic cations can be within the specific range from about 4 mM to about 9 mM and more preferably, within the range from about 5 mM to about 7 mM. 
     The reaction mixture may include endogenous NTPs (i.e., NTPs that are present in the cell extract) and or exogenous NTPs (i.e., NTPs that are added to the reaction mixture). In certain aspects, the reaction use ATP, GTP, CTP, and UTP. In certain aspects, the concentration of individual NTPs is within the range from about 0.1 mM to about 2 mM. 
     The reaction mixture may include any alcohol suitable for CFPS. In certain aspects, the alcohol may be a polyol, and more specifically glycerol. In certain aspects the alcohol is between the general range from about 0% (v/v) to about 25% (v/v), including specific intermediate values of about 5% (v/v), about 10% (v/v) and about 15% (v/v), and about 20% (v/v), among others. 
     The components, systems, and methods disclosed herein may be applied to recombinant cell systems and cell-free protein synthesis methods in order to prepare glycosylated proteins. Glycosylated proteins that may be prepared using the disclosed components, systems, and methods may include proteins having N-linked glycosylation (i.e., glycans attached to nitrogen of asparagine). The glycosylated proteins disclosed herein may include unbranched and/or branched sugar chains composed of monomers as known in the art such as glucose (e.g., β-D-glucose), galactose (e.g., β-D-galactose), mannose (e.g., β-D-mannose), fucose (e.g., α-L-fucose), N-acetyl-glucosamine (GlcNAc), N-acetyl-galactosamine (GalNAc), neuraminic acid, N-acetylneuraminic acid (i.e., sialic acid), and xylose, which may be attached to the glycosylated proteins, growing glycan chain, or donor molecule (e.g., a sugar donor nucleotide) via respective glycosyltransferases (e.g., N-glycosyltransferases). The glycosylated proteins disclosed herein may include glycans as known in the art including but not limited to Man 3 GlcNAc 2  glycan, Man 5 GlcNAc 3  glycan, and the fully sialylated human glycan Man 3 GlcNAc 4 Gal 2 Neu 5 Ac 2 . 
     In certain exemplary embodiments, one or more of the methods described herein are performed in a vessel, e.g., a single, vessel. The term “vessel,” as used herein, refers to any container suitable for holding on or more of the reactants (e.g., for use in one or more transcription, translation, and/or glycosylation steps) described herein. Examples of vessels include, but are not limited to, a microtitre plate, a test tube, a microfuge tube, a beaker, a flask, a multi-well plate, a cuvette, a flow system, a microfiber, a microscope slide and the like. 
     Glycosylation in Prokaryotes 
     Glycosylation in prokaryotes is known in the art. (See e.g., U.S. Pat. Nos. 8,703,471; and 8,999,668; and U.S. Published Application Nos. 2005/0170452; 2006/0211085; 2006/0234345; 2006/0252672; 2006/0257399; 2006/0286637; 2007/0026485; 2007/0178551; and International Published Applications WO2003/056914A1; WO2004/035605A2; WO2006/102652A2; WO2006/119987A2; and WO2007/120932A2; the contents of which are incorporated herein by reference in their entireties). 
     Self-Assembled Monolayers for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (SAMDI-MS) 
     The disclosed methods may utilize self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS), for example, as a method for detecting glycosylation of peptides and proteins in the disclosed methods and systems. SAMDI-MS is known in the art and has been utilized to study peptides, proteins, and carbohydrates and their reaction products. (See Ban et al., “Discovery of Glycosyltransferases Using Carbohydrate Arrays and Mass Spectrometry,” Nat. Chem. Biol., 2012, 8, 769-773; Ban et al., “On-Chip Synthesis and Label-Free Assays of Oligosaccharide Arrays,” Chem. Int. Ed., 2008, 47(18), 3396-3399; Houseman et al., “Maleimide-Functionalized Self-Assembled Monolayers for the Preparation of Peptide and Carbohydrate Biochips,” Langmuir, 2003, 19(5), 1522-1531; Su et al., “Using Mass Spectrometry to Characterize Self-Assembled Monolayers Presenting Peptides, Proteins and Carbohydrates,” Angew. Chem. Int. Ed., 2002, 41, 4715-4718; Houseman et al., “Toward Quantitative Assays with Peptide Chips: A Surface Engineering Approach,” Trends Biotech., 2002, 20 (7), 279-281; Houseman et al., “Carbohydrate Arrays for the Evaluation of Protein Binding and Enzyme Activity,” Chem. Biol., 2002, 9, 443-454); and Laurent, N., et al. (2008). “Enzymatic Glycosylation of Peptide Arrays on Gold Surfaces.” Chembiochem 9(6): 883-887); the contents of which are incorporated herein by reference in their entireties). 
     Miscellaneous 
     The steps of the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The steps may be repeated or reiterated any number of times to achieve a desired goal unless otherwise indicated herein or otherwise clearly contradicted by context. 
     Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 
     ILLUSTRATIVE EMBODIMENTS 
     The following embodiments are illustrative and are not intended to limit the scope of the claimed subject matter. 
     Embodiment 1. A method for synthesizing a glycoprotein or a recombinant glycoprotein, the method comprising: (a) expressing in a cell or in a cell-free protein synthesis (CFPS) reaction a polypeptide comprising the amino acid sequence of a target protein which naturally includes an amino acid motif optionally comprising at least about four (4) amino acids including an asparagine that is glycosylated by an N-glycosyltransferase or that has been modified to include a heterologous amino acid motif optionally comprising at least about four (4) amino acids including an asparagine that is glycosylated by an N-glycosyltransferase, the amino acid motif or heterologous amino acid motif optionally comprising an amino acid sequence selected from SEQ ID NOs:1-549; (b) expressing in a cell or in a CFPS reaction the N-glycosyltransferase; and (c) reacting the polypeptide and the N-glycosyltransferase in the presence of a sugar donor, wherein the N-glycosyltransferase glycosylates the amino acid motif or the heterologous amino acid motif of the polypeptide with the sugar to synthesize the glycoprotein or recombinant glycoprotein (optionally wherein step (a) comprises expressing in a cell or in a cell-free protein synthesis (CFPS) reaction a polypeptide comprising the amino acid sequence of a target protein which has been modified to include two or more different heterologous amino acid motifs that includes an asparagine that is glycosylated by two or more different N-glycosyltransferases, the heterologous amino acid motifs optionally comprising an amino acid sequence selected from SEQ ID NOs:1-549; and optionally wherein step (b) comprises expressing in one or more cells or in one or more CFPS reactions the two or more different N-glycosyltransferases, wherein the two or more different N-glycosyltransferases are expressed simultaneously in the same cell or CFPS reaction or sequentially in two or more different cells or two or more different CFPS reactions; and optionally wherein step (c) comprises reacting the polypeptide and the two or more different N-glycosyltransferases in the presence of two or more sugar donors which are the same or different, wherein the polypeptide is reacted with the two or more different N-glycosyltransferases simultaneously for example in the same cell or CFPS reaction, or sequentially for example in two or more different cells or two or more different CFPS reactions, and wherein the two or more different N-glycosyltransferases glycosylate the two or more different heterologous amino acid motifs of the polypeptide with the sugar of the two or more sugar donors to synthesize the glycoprotein or recombinant glycoprotein). 
     Embodiment 2. The method of embodiment 1, wherein the amino acid motif or heterologous amino acid motif comprises a sequence X −2 -X −1 -N-X +1 -S/T-X +3 , wherein X −2  is selected from Gly, Asn, and Tyr; X −1  is selected from Gly and Ala; X +1  is selected from Trp, Val, His, Ala, and Be; and X +3  is selected from Thr, Met, and Phe. 
     Embodiment 3. The method of embodiment 1, wherein the target protein is a eukaryotic protein. 
     Embodiment 4. The method of embodiment 1, wherein the target protein is a prokaryotic protein. 
     Embodiment 5. The method of any of embodiments 1-4, wherein step (a) is performed in a prokaryotic cell. 
     Embodiment 6. The method of any of embodiments 1-4, wherein step (b) is performed in a prokaryotic cell. 
     Embodiment 7. The method of any of embodiments 1-4, wherein step (a) and step (b) are performed in the same prokaryotic cell. 
     Embodiment 8. The method of any of embodiments 1-4, wherein step (a) is performed in a eukaryotic cell. 
     Embodiment 9. The method of any of embodiments 1-4, wherein step (b) is performed in a eukaryotic cell. 
     Embodiment 10. The method of any of embodiments 1-4, wherein step (a) and step (b) are performed in the same eukaryotic cell. 
     Embodiment 11. The method of any of embodiments 1-4, wherein step (a) is performed in a prokaryotic-based CFPS reaction. 
     Embodiment 12. The method of any of embodiments 1-4, wherein step (b) is performed in a prokaryotic-based CFPS reaction. 
     Embodiment 13. The method of any of embodiments 1-4, wherein step (a) and step (b) are performed in the same prokaryotic-based CFPS reaction. 
     Embodiment 14. The method of any of embodiments 11-13, wherein step (c) is performed in the same prokaryotic-based CFPS reaction as step (a) and/or step (b). 
     Embodiment 15. The method of any of the foregoing embodiments, wherein the N-glycosyltransferase is a prokaryotic N-glycosyltransferase. 
     Embodiment 16. The method of embodiment 15, wherein the prokaryotic N-glycosyltransferase is a prokaryotic N-glycosyltransferase from one of  Actinobacillus  spp.,  Escherichia  spp.,  Haemophilus  spp., or  Mannheimia  spp. 
     Embodiment 17. The method of embodiment 15, wherein the prokaryotic N-glycosyltransferase is a prokaryotic N-glycosyltransferase from one of  Actinobacillus pleuropneumoniae, Escherichia coli, Haemophilus  influenza,  Mannheimia haemolytica , or  Haemophilus dureyi.    
     Embodiment 18. The method of any of embodiments 1-17, wherein multiple distinct and/or non-naturally occurring glycans are introduced to a protein by specifically choosing unique sequence:enzyme pairs that allow for orthogonal, and/or parallel and/or independent glycosylation. 
     Embodiment 19. The method of any of embodiments 1-18 further comprising immobilizing the polypeptide (e.g., covalently immobilizing the polypeptide) on a solid support (e.g., magnetic beads) prior to performing one or more of step (a), step (b), and/or step (c), wherein the polypeptide may be immobilized directly to the solid support or indirectly to the solid support via a linking moiety (e.g., a covalently linking protein or peptide). 
     Embodiment 20. A method for synthesizing a glycoprotein or recombinant glycoprotein, the method comprising: (a) expressing in a cell or in a cell-free protein synthesis (CFPS) reaction a polypeptide comprising the amino acid sequence of a target protein which includes naturally two or more different amino acid motifs that includes an asparagine that is glycosylated by two or more different N-glycosyltransferases or that has been modified to include two or more different heterologous amino acid motifs that includes an asparagine that is glycosylated by two or more different N-glycosyltransferases, the amino acid motifs or heterologous amino acid motifs optionally comprising an amino acid sequence selected from SEQ ID NOs:1-549; (b) expressing in one or more cells or in one or more CFPS reactions the two or more different N-glycosyltransferases, wherein the two or more different N-glycosyltransferases are expressed simultaneously in the same cell or CFPS reaction or sequentially in two or more different cells or two or more different CFPS reactions; and (c) reacting the polypeptide and the two or more different N-glycosyltransferases in the presence of two or more sugar donors which are the same or different, wherein the polypeptide is reacted with the two or more different N-glycosyltransferases simultaneously for example in the same cell or CFPS reaction, or sequentially for example in two or more different cells or two or more different CFPS reactions, and wherein the two or more different N-glycosyltransferases glycosylate the two or more different amino acid motifs or heterologous amino acid motifs of the polypeptide with the sugar of the two or more sugar donors to synthesize the glycoprotein or recombinant glycoprotein; optionally wherein the method comprises sequentially conjugating monosaccharides to the polypeptide by reacting the two or more different N-glycosyltranferases with the polypeptide wherein the two or more different N-glycosyltranferases transfer single or multiple saccharides to the polypeptide at different positions and produce different glycosylation structures at the different positions. 
     Embodiment 21. The method of embodiment 20, further comprising immobilizing the polypeptide (e.g., covalently immobilizing the polypeptide) on a solid support (e.g., magnetic beads) prior to performing one or more of step (a), step (b), and/or step (c); and optionally washing the polypeptide after performing one or more of step (a), step (b), and/or step (c) (for example after a glycosylation step); and optionally releasing the polypeptide from the solid support (for example, via treatment with a protease) after performing one or more of step (a), step (b), and/or step (c), wherein the polypeptide may be immobilized directly to the solid support or indirectly to the solid support via a linking moiety (e.g., covalently linking protein or peptide). 
     Embodiment 22. A method for selecting an amino acid motif that is glycosylated by an N-glycosyltransferase, the method comprising: (a) reacting a library of peptides with an N-glycosyltransferase in the presence of a sugar donor, wherein the N-glycosyltransferase glycosylates one or more of the peptides; (b) detecting glycosylation of the reacted peptides by immobilizing the reacted peptides on a substrate comprising self-assembled monolayers, and performing matrix-assisted laser desorption/ionization mass spectrometry of the immobilized reacted peptides to select the amino acid motif that is glycosylated by the N-glycosyltransferase. 
     Embodiment 23. The method of embodiment 22, wherein the library comprises at least about 10, 50, 100, 500, or 1000 peptides. 
     Embodiment 24. The method of embodiment 22 or 23, wherein the peptides comprise at least 6 amino acids and have a sequence X −2 -X −1 -N-X +1 -S/T-R-C wherein X is any amino acid. 
     Embodiment 25. The method of any of embodiments 22-24, wherein the peptides are covalently immobilized on the substrate comprising the self-assembled monolayers. 
     Embodiment 26. The method of embodiment 25, wherein the library of peptides comprise a C-terminal Cys, the self-assembled monolayers comprise free maleimides, and the C-terminal Cys of the peptides reacts with the free maleimides to form a bond (e.g., a C—S bond) and covalently immobilize the peptide. 
     Embodiment 27. The method of embodiment 26, wherein the self-assembled monolayers comprise alkylthiolates which provide the free maleimides. 
     Embodiment 28. The method of embodiment 25, wherein: (i) the library of peptides comprise a C-terminal alkyne, the self-assembled monolayers comprise free azides, and the C-terminal alkyne of the peptides reacts with the free azides to form a bond (e.g., a triazole and in particular a 1,2,3-triazole) and covalently immobilize the peptide; or (ii) the library of peptides comprise a C-terminal azide, the self-assembled monolayers comprise free alkynes, and the C-terminal azide of the peptides reacts with the free alkynes to form a bond (e.g., a triazole and in particular a 1,2,3-triazole) and covalently immobilize the peptide. 
     EXAMPLES 
     The following Examples are illustrative and are not intended to limit the scope of the claimed subject matter. 
     Example 1—Design of Protein Sequence for Cytoplasmic Glycosylation 
     Abstract 
     Glycosylation modulates the pharmacokinetics and potency of protein therapeutics and vaccines. However, current methods cannot sufficiently explore the vast experimental landscapes required to accurately predict and design glycosylation sites for specific glycosyltransferases (GTs). We used a systematic platform for Rapid Expression and Characterization to Direct Efficient Glycosylation (RECoDE-G) using Cell-Free Protein Synthesis (CFPS) and Self-Assembled Monolayers for Desorption Ionization Mass Spectrometry (SAMDI-MS) to produced &gt;500 μg/mL of five cytoplasmic N-linked glycosyltransferases (NGTs) in vitro and determine their peptide acceptor and sugar donor specificities at unprecedented depth and throughput with ˜3,000 unique peptides and ˜10,000 unique reaction conditions. We found that peptide selectivity data closely matched glycosylation trends on small sequon motifs (GlycTags) within heterologous proteins, including an Fc human antibody fragment, in vitro and in the cytoplasm of living cells. The data collected in this work allows for design of polypeptide sequences for efficient modification with NGTs and the RECoDE-G workflow provides a systematic tool to characterize other polypeptide glycosyltransferases. 
     Applications 
     The applications of the disclosed technology may include, but are not limited to: (i) design of therapeutic polypeptide amino acid sequences for improved glycosylation by an N-linked glycosyltransferase in vitro or in a cell; (ii) high-throughput characterization of glycosyltransferases peptide specificities or engineering of glycosyltransferases for alternative peptide or sugar specificities; and (iii) production of high titers of proteins in industrial bacterial host organisms which are glycosylated site-specifically in the bacterial cytoplasm. 
     Advantages 
     The advantages of the disclosed technology may include, but are not limited to: (i) NGT glycosylation systems allow for efficient modification of polypeptides without a eukaryotic host or lipid-bound substrates and enzymes, where previously this system was only functional on autotransporter protein substrates or long, repeated glycosylation sequences, and our findings allow for informed design of glycosylation sites within polypeptides for efficient modification in vitro or in living cells; (ii) demonstrated first glycosylation of human IgG Fc fragment in  E. coli  cytoplasm using redesigned sequences which direct efficient glycosylation by NGT; (iii) the ability to design glycosylation sites for modification with NGT allows for the production of glycoproteins in the bacterial cytoplasm, obviating the need for transport to the bacterial periplasm (as is required to use existing oligosaccharyltransferase glycosylation methods); (iv) this innovation will increase the diversity of glycoproteins which can be produced in bacteria, a preferred industrial host strain; and (v) the use of the SAMDI method allows for the rapid study of 1000&#39;s of peptides across multiple enzymes and 10,000&#39;s of reaction conditions, where in contrast, current studies of glycosyltransferase specificity require expression and purification of the enzyme from cells by affinity purification, screening by incorporation of radioactively or chemically labeled sugars or liquid chromatography (LC) methods, and validation by mass spectrometry (typically LC-MS), and these current methods limit investigations to 10-100 peptides. 
     Description of Technology 
     Most methods for glycoprotein synthesis use eukaryotic organisms. Bacterial glycosylation offers the opportunity to more closely control glycosylation patterns and more rapidly develop more diverse glycosylation systems. Most existing methods use a membrane bound oligosaccharyltransferase (OST) to transfer lipid-linked sugar donors en bloc onto proteins. 
     NGTs are soluble enzymes which transfer sugars from activated donors directly onto proteins without the use of membrane bound components. However, their use for the modification of heterologous proteins has been limited, likely due to an incomplete understanding of peptide specificity and therefore an inability to design efficiently modified glycosylation sites. 
     Two studies by Naegali et al. in 2014 attempted to characterize the sequence specificity of NGT by directly measuring modification of ˜10 peptides by HPLC analysis and by LC-MS/MS of  E. coli  cells in which ApNGT was expressed. (See Naegeli, A. et al., “Substrate Specificity of Cytoplasmic N-Glycosyltransferase,”  Journal of Biological Chemistry  289, 24521-24532 (2014); and Naegeli, A. et al., “Molecular analysis of an alternative N-glycosylation machinery by functional transfer from  Actinobacillus pleuropneumoniae  to  Escherichia coli,” The Journal of biological chemistry  289, 2170-2179 (2014)). These studies showed that NGT can efficiently modify some N-X-S/T motifs with glucose, galactose, xylose, or mannose and showed trends of modification in living cells. This study also showed that ApNGT can modify wildtype human erythropoietin in the  E. coli  cytoplasm (although protein solubility and glycosylation efficiency was not determined). Other work by the Aebi lab disclosed in a patent showed modification of wildtype bacterial autotransporter proteins (native substrates for NGTs) in cells and their potential use as a vaccine. (See Schwarz, et al, “Cytoplasmic N-Glycosyltransferase of  Actinobacillus pleuropneumoniae  Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence,”  Journal of Biological Chemistry  286, 35267-35274 (2011)). 
     In 2017, Cuccui et al. achieved glycosylation in cells with NGT by fusing 12 repeating glycosylation sites to the C-terminus of a bacterial protein (no data was provided on glycosylation efficiency or if this glycosylation was due to native sites within the protein rather than the added sites). (See Cuccui, J. et al., “The N-linking glycosylation system from  Actinobacillus pleuropneumoniae  is required for adhesion and has potential use in glycoengineering,”  Open biology  7 (2017)). Another 2017 study by Song et al. developed an ApNGT variant with improved activity and wider peptide specificity. (See Song et al., “Production of homogeneous glycoprotein with multi-site modifications by an engineered N-glycosyltransferase mutant,”  Journal of Biological Chemistry  (2017)). In this study, peptide specificity of wildtype ApNGT and the engineered variant was characterized using ˜40 peptides and the sequence of the bacterial autotransporter protein HMW1 was altered to improve its glycosylation in vitro. The scope of our SAMDI study of peptide specificity characterization exceeds this work by an order of magnitude. Efficiency of glycosylation of HMW1 was not reported site-by-site but rather in aggregate, leading to uncertainties in the direct effect of modifying each site. Furthermore, the breadth of our specificity data enabled us to redesign glycosylation sites into diverse proteins not usually modified by NGT including a therapeutically relevant human IgG protein in cells and a protein which previously did not contain glycosylation sites ( E. coli  protein Im7). 
     Our findings allow for site-specific and efficient enzymatic N-linked glycosylation of diverse proteins in vitro and in the bacterial cytoplasm by design of primary amino acid sequences. This technique could enable quicken development and reduce production costs for glycoprotein therapeutics. The method we developed using SAMDI-MS and CFPS can rapidly recapitulate these results for other enzymes homologs or enzyme variants of interest. 
     This technology also allows for the production of site-specifically glycosylated proteins, including protein therapeutics and vaccines. The lipid-independent nature of this system makes it attractive for in vitro modification of protein therapeutics and glycosylation in the bacterial cytoplasm. These high-titer, rapid expression systems could allow glycoprotein therapeutics to be developed and produced more quickly and at lower cost. 
     Example 2—Design of Glycosylation Sites by Rapid Expression and High-Throughput Characterization of N-glycosyltransferases 
     Reference is made to the manuscript entitled “Design of glycosylation sites by rapid expression and high-throughput characterization of N-glycosyltransferase,” by Weston Kightlinger, Liang Lin, Madisen Rosztoczy, Matthew P. DeLisa, Milan Mrksich, and Michael C. Jewett, Nat. Chem. Biol., 2018 May 7, doi: 10.1038/s41589-018-0051-2, which content is incorporated herein by reference in its entirety. 
     Abstract 
     Glycosylation is an abundant post-translational modification that is important in disease and biotechnology. Current methods to understand and engineer glycosylation cannot sufficiently explore the vast experimental landscapes required to accurately predict and design glycosylation sites modified by glycosyltransferases. Here we describe a systematic platform for glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES), which combines cell-free protein synthesis and mass spectrometry of self-assembled monolayers. We produced six N- and O-linked polypeptide-modifying glycosyltransferases from bacteria and humans in vitro and rigorously determined their substrate specificities using 3,480 unique peptides and 13,903 unique reaction conditions. We then used GlycoSCORES to optimize and design small glycosylation sequence motifs that directed efficient, N-linked glycosylation in vitro and in the  Escherichia coli  cytoplasm for three heterologous proteins, including human immunoglobulin Fc domain. We find that GlycoSCORES is a broadly applicable method to facilitate fundamental understanding of glycosyltransferases and engineer synthetic glycoproteins. 
     Introduction 
     Protein glycosylation is the post-translational attachment of complex oligosaccharides (glycans), most commonly at asparagine (N-linked) or serine and threonine (O-linked) amino acid side chains 1, 2 . Glycosylation is found in all domains of life and plays critical roles in cellular function 2 . Glycosylation is also present in 70% of approved or preclinical protein therapeutics 3  and has profound effects on protein stability, immunogenicity 5 , and potency 6 , motivating close study and intentional engineering of glycosylation sites and structures 7 . Production of glycoproteins within native hosts often results in structural heterogeneity, limits titers and genetic tractability, and constrains the diversity of glycans that can be produced 8-10 . These difficulties have motivated the development of highly-engineered glycosylation systems within mammalian cells 11 , yeast 12 , bacteria 8, 9 , and in vitro 6, 13  to produce more homogeneous human-like glycans for therapeutics 12 , bacterial glycans for vaccines 9 , and synthetic glycans for fundamental biology studies 6, 10 . 
     Despite these advances, major glycoengineering challenges and gaps in understanding of natural glycosylation systems still remain due, in large part, to a lack of high-throughput methods for synthesis and detailed biochemical characterization of glycosyltransferases (GTs), the enzymes that attach and elaborate glycans on proteins. GTs are the catalytic nodes of natural systems and the parts from which synthetic glycosylation systems are constructed; and yet, less than 1% of putative GTs have been biochemically characterized 14  with far fewer at sufficient depth to be useful in biocatalysis 15 . Typically, studies of GT specificity require expression and purification of the enzyme from cells; screening by incorporation of radioactively or chemically labeled sugars 16, 17 , antibody detection 17, 18 , or liquid chromatography (LC) separation 19 ; and validation by mass spectrometry (usually LC-MS/MS)?. Existing methods are particularly problematic for characterizing GTs that attach glycans to polypeptides. These polypeptide GTs (ppGTs) include the O-linked polypeptide N-acetylgalactosaminyltransferase (ppGalNAcT), O-linked N-acetylglucosamine transferase (OGT), and oligosaccharyltransferase (OST) enzyme families. Such enzymes are of particular interest because they determine which sites on a protein are glycosylated and constrain the possible glycoforms that can be installed. A recently discovered ppGT called N-glycosyltransferase from the bacterial pathogen  Actinobacillus pleuropneumoniae  (NGT), has elicited a great deal of interest for biocatalysis 9, 21, 22  because it is a soluble, cytoplasmic enzyme which can efficiently install an N-linked glucose on N-X-S/T amino acid sequence motifs resembling those in eukaryotic proteins using uracil-diphosphate-glucose (UDP-Glc) as a sugar donor 23 . While pioneering efforts by several groups reported several protein and peptide substrates that can be modified by NGT 9, 20, 24-27 , current methods for GT analysis limits investigations of NGT and also other ppGTs to only dozens of unique peptide substrates. These methods undersample the vast amino acid sequence space available for modification, providing incomplete information of amino acid preferences at each position surrounding the glycosylation site and the interdependency of amino acids at these positions, which are required for a full understanding of GTs in natural systems and for the rational design of efficient protein glycosylation sites. 
     Here we report a generalizable and systematic strategy for glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES). GlycoSCORES couples expression by  Escherichia coli -based cell-free protein synthesis (CFPS) to functional characterization of GTs with self-assembled monolayers for matrix-assisted desorption/ionization (SAMDI) mass spectrometry. This workflow enables high-throughput, label-free, quantitative analysis of peptide glycosylation without time-consuming cell lysis and protein purification. We apply the GlycoSCORES workflow to the study of NGT, two previously uncharacterized NGT homologs, namely human ppGalNAcT1 and ppGalNAcT2, and human OGT (hOGT) using 3,480 unique acceptor peptides and 13,903 unique reaction conditions. We demonstrate the utility of GlycoSCORES for glycoprotein engineering by rigorously optimizing NGT acceptor sequences to inform the design of improved glycosylation sites. We identify several small glycosylation tag sequence motifs termed “GlycTags” (originally described by Imperiali 28 , DeLisa 29 , and others) and used them to direct efficient glycosylation of several target proteins including the  E. coli  immunity protein Im7, the  H. influenzae  autotransporter protein (HMW1ct) and the constant region (Fc) of a human immunoglobulin (IgG1) antibody. We find that glycosylation efficiencies of GlycTag sequences within proteins closely mirrored trends observed from GlycoSCORES analysis of peptides. Upon synthesis and glycosylation by NGT in the cytoplasm of living  E. coli , proteins glycosylation sites that were redesigned according to a GlycoSCORES-derived GlycTag sequence were modified more efficiently than naturally occurring glycosylation sequences and a previously identified NGT glycosylation consensus sequence 20 . 
     Results 
     Development of GlycoSCORES for characterization of NGT. We selected NGT as the primary GT model to demonstrate the GlycoSCORES framework ( FIG. 1 ) because of its potential for biocatalysis and our hypothesis that a deeper analysis of the NGT acceptor substrate sequence space would enable the rational redesign of protein glycosylation sites for improved modification by NGT. Because difficulties in protein expression and purification are key challenges for GT characterization, we chose to use  E. coli -based crude lysate CFPS for GT expression in the GlycoSCORES workflow. CFPS can rapidly produce g/L quantities of many complex proteins, is compatible with liquid handling robotics for direct coupling to our SAMDI analysis pipeline, and allows for enzyme quantification and functional analysis without cell lysis, affinity tags, or purification 30   . E. coli  lysates also lack native protein glycosylation activity, providing a blank canvas for bottom-up engineering and characterization. CFPS reactions were used to express soluble NGT at 20° C. for 20 h, at which point protein synthesis was &gt;95% complete ( FIG. 7  and data not shown). Soluble NGT was quantified as 814±97 μg/mL by  14 C-leucine incorporation and visualized by SDS-PAGE autoradiogram ( FIG. 8 ). 
     We next developed a SAMDI method for high-throughput analysis of NGT peptide specificity. The SAMDI method uses alkanethiolate self-assembled monolayers (SAMs) to capture enzyme reaction substrates and products, which are purified on-chip, detected, and quantified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We previously demonstrated that the SAMDI assay could be used to profile the substrate specificities of several enzyme classes including deacetylases 31 , acetyltransferases 32 , and DNA ligases 33 . We also reported SAMDI screening of GT activities using monolayers presenting 24 immobilized sugars 15 . Previous works 34, 35  by the Sabine group have also reported the activity of the polypeptide N-acetylgalactosylamine transferase 2 (ppGalNAcT2) on immobilized peptides, though with limited substrate numbers. Here, we synthesized peptide sequence libraries for testing NGT activity by solid-phase peptide synthesis (SPPS) with a C-terminal Cys residue for specific immobilization via a Michael-Addition reaction onto SAMs that present maleimide groups against a background of tri(ethylene glycol) groups ( FIG. 2 a   ). We then performed in vitro glycosylation (IVG) reactions by adding NGT and UDP-Glc sugar donor to peptide libraries. Completed IVG reactions were transferred onto SAMDI plates for immobilization onto 384 maleimide SAM spots followed by rinsing and MALDI-MS of the SAMs, which provided distinct mass peaks for alkyldisulfides terminated in unmodified and glycosylated peptides. Integration of these peaks (with adjustments for ionization suppression due to glycosylation, see Methods, and data not shown) provided the glycosylation efficiency of each reaction ( FIG. 2 a   ). We validated this approach using NGT purified from BL21 (DE3)  E. coli  (data not shown) and truncated versions of the previously used NGT peptide substrate GSDQNATF 19 . We observed the appearance of a new MS signal at +162 m/z (the mass of a glucose moiety) in peptides as short as CNAT and NATC ( FIG. 9 ). 
     Using the methods above, we then created the complete GlycoSCORES platform using NGT synthesized in crude lysate CFPS reactions to glycosylate peptides in solution, which are then captured on-chip and directly analyzed by SAMDI ( FIG. 2 a   ). We evaluated the activity of NGT produced in CFPS against a peptide library having the sequence X −1 NX +I TRC where X +1  and X −1  are one of 19 amino acids (Cys is excluded) ( FIG. 2 b    and data not shown). The glycosylation efficiency of these peptide sequences varied from no observed activity to nearly 100% conversion. NGT preferred Pro and Ala at the X −1  position and Val, His, Ala, and Ile at the X +1  position. Low modification was observed for Lys and Arg (basic residues) at the X −1  position, Pro (a conformationally constrained residue) at the X +1  position, and Asp and Glu (acidic residues) at the X +1  position ( FIG. 2 b   ). Our results extend recent findings that charged amino acids adjacent to the NGT modification site are generally disfavored 24  by showing that positively charged residues are most disfavored in the X +1  position while negatively charged residues are most disfavored in the X −1  position. 
     Interestingly, the preference of NGT for a given amino acid at a given sequence position is dependent on nearby amino acids. For example, Trp in the X +1  position is generally not well tolerated, however, the motif GNWTRC was among the most efficiently glycosylated sequences ( FIG. 2 b   ). These unexpected dependencies necessitate the sampling of large, combinatorial libraries, which requires a high-throughput assay such as GlycoSCORES. Importantly, NGT produced in CFPS showed similar peptide selectivity and specific activity to purified NGT across the X −1 NX +1 TRC library ( FIG. 10 ). Control IVG reactions performed with CFPS reactions synthesizing superfolder green fluorescent protein (sfGFP) rather than NGT showed no peptide modification (data not shown). 
     While we focused on peptide specificity, we also showed the breadth of the platform by screening NGT activity with 6 different nucleotide-activated sugar donors: UDP-Glc, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), guanosine-diphosphate mannose (GDP-Man), and UDP-xylose (UDP-Xyl). Consistent with previous results 19 , we found that NGT transferred UDP-Glc with the highest efficiency and UDP-Gal and UDP-Xyl with much lower efficiencies ( FIG. 11 ). We also tested the interdependency of sugar donor on the X −1 NX +1 T peptide library selectivity. While the peptide selectivity remained similar with each of the UDP-Glc, UDP-Gal, and UDP-Xyl donors, sugar donor identity did influence relative X −1  and X +1  residue preferences in some cases. For example, NGT is less tolerant of Trp at the X −1  position when transferring xylose compared to glucose or galactose. 
     Study of NGT homolog and human O-linked GT peptide specificities. To demonstrate the utility of GlycoSCORES for analysis of uncharacterized glycosyltransferases, we synthesized NGT homologs from the bacterial pathogens  Mannheimia haemolytica  and  Haemophilus ducreyi  (MhNGT and HdNGT), found that they are in fact NGTs, and determined their specificity on all possible acceptor sequences within the canonical NGT target sequence of X −1 -N-X +1 -S/T ( FIG. 12  and data not shown). Given sequence identities of 68-76% ( FIG. 12 ), these enzymes show striking similarities to NGT acceptor sequence preferences. This may indicate two-fold evolutionary pressure for these enzymes to modify designated target proteins, but not modify and interfere with essential cytoplasmic proteins. The discovery of NGTs with conserved specificities in these organisms motivates further studies to understand the roles these enzymes in their pathogenesis. 
     We also applied the GlycoSCORES workflow to determine the peptide specificities of two important O-linked human glycosylation enzymes, ppGalNAcT1 and ppGalNAcT2. These enzymes install the first sugar of mucin-like glycans which effect the development of several cancers- and aberrant lipid metabolism-. Both ppGalNAcT1 and ppGalNAcT2 were produced in CFPS (data not shown) and characterized with a saturated X −1 -T-X +1 -P peptide library ( FIG. 3  and  FIG. 13 ). In addition to corroborating previous investigations of the specificity of these enzymes on peptides in vitro 34  and on proteins in vivo 37, 41 , the throughput of GlycoSCORES allowed us to simultaneously vary both the X −1  and X +1  positions, which was difficult using conventional strategies, and obtain quantitative readouts for each combination. Our data led to unexpected results in the specificities of ppGalNAcT1 and ppGalNAc2. For example, we discovered that intolerance of aromatic residues adjacent to the glycosylation site of ppGalNAcT2 can be overcome by the presence of a Pro in the X −1  position. However, the preference of ppGalNAcT2 for Pro is reduced in the cases of Val and Thr in the X +1  position, which themselves would predict relatively good substrates ( FIG. 3 ). The ability to investigate ppGalNAcT specificity at this depth could advance efforts to design of isoform-specific substrates and substrate mimics and enhance glycosylation site prediction. 
     We further demonstrated the broad applicability of GlycoSCORES by characterizing the human O-linked GlcNAc transferase (hOGT), which has been implicated in neurodegeneration and insulin resistance disease states 42, 43 . We expressed hOGT in CFPS (Supplementary  FIG. 14 ) and analyzed 19-amino acid substitutions for each position of a proposed OGT targeting sequence of PPVSR 16  ( FIG. 14 ). Specificity results were largely consistent with previous analysis of human glycosylation sites and peptide screens 16, 44-46 , such as the preference for V in the X 3  position and A in the X +2  position. When we extended the sequence to the X −4  and X +2  position, we discovered an overall increase in modification and a strong preference for aromatic residues. This screen provides proof of concept for future studies of OGT variants, which can be rapidly produced in CFPS and probed for peptide specificity at high depth and throughput. 
     Optimization of NGT peptide acceptor sequences. To show that GlycoSCORES can be used for rigorous acceptor peptide sequence optimization towards increased modification of whole proteins, we sought to develop optimized GlycTag sequences for NGT comprised of 6 amino acids that could be efficiently modified in the context of whole proteins. Because the total number of 6-mer sequences is prohibitively large, we iteratively tested preferences in the X +2 , X −2 , and X +3  positions across a set of sequences informed by previous libraries ( FIG. 4  and data not shown). To determine the preference of NGT for amino acids at the X +2  position, we prepared an array of 380 peptides based on the motif (X −1 NX +1 )X +2 RC, by selecting 20 efficiently glycosylated X −1 NX +1 T sequences from  FIG. 2 b    and resynthesizing each with 19 amino acids in the X +2  position ( FIG. 4 a   ). As was previously reported 19, 24 , we found Thr or Ser are required in the X +2  position for efficient modification by NGT, with Thr preferred over Ser. We also evaluated a complete X −1 NX +1 S peptide library and did not find significant differences in X −1  and X +1  residue preferences compared to the X −1 NX +1 T peptide library ( FIG. 15 ). 
     We next evaluated NGT activity for X −2  residues using 40 sequences selected from the X −1 NX +1 X +2 RC screens shown in  FIG. 2 b    and  FIG. 4 a   . To allow for synergistic and compensatory mutations leading to overall greater activity in later screens, we balanced preservation of sequence diversity while narrowing our search towards an optimized sequence. This narrowing approach is justified by the observation that sequence modification trends generally matched those predicted by earlier screens (see blue, grey, and black boxes and text in  FIGS. 2 b    and  4 ). We resynthesized the 40 sequences from  FIG. 2 b    and  FIG. 4 a    with 19 amino acids in the X −2  position and performed GlycoSCORES characterization of a 760-peptide library of the form X −2 (X −1 NX +1 X +2 )RC ( FIG. 4 b   ). We found that Gly, Asn, and Tyr were preferred at the X −2  position while Lys gave low activity. This library showed robust modification of the GNWT motif even when a non-preferred residue was present in the X −2  position. While Pro was a preferred residue in the X −1  position of the X −1 NX +1 X +2 RC libraries ( FIGS. 2 b  and 4 a    and data not shown), we found that most of these sequences were poorly modified with the addition of an X −2  residue, especially when the X −2  residue was Asn ( FIG. 4 b    and data not shown). This effect is likely related to the conformational constraints of Pro. 
     Finally, we synthesized and evaluated a library of 1140 peptides having the sequence (X −2 X −1 NX +1 X +2 )X +3 RC with 19 amino acids in the X +3  position ( FIG. 4 c   ) based on 60 sequences selected from  FIG. 4 b   . We discovered efficient glycosylation of peptides containing Thr, Met, and Phe in the X +3  position, but not peptides with Pro, Asp, and Glu at this position. We found that 59 acceptor peptides had greater than 70% modification efficiency after exposure to just 0.025 μM NGT for 1 h (data not shown). We chose one efficiently modified 6-mer sequence (YANATT (SEQ ID NO:518)) to test NGT preference for the residue that undergoes glycosylation. We found that only peptides with Asn at the X 0  position showed detectable modification ( FIG. 16 ). 
     GlycTags enable efficient protein glycosylation in vitro. From the peptide screens described above, we hypothesized that preferred peptide substrates found by GlycoSCORES could be glycosylated by NGT when engineered into whole proteins. We chose the sequence GGNWTT (SEQ ID NO:514) as a model because it was found to be more efficiently glycosylated than any previously studied 19, 20, 23, 47  6-mer sequences that we tested ( FIG. 17 ). The preference of NGT for the GGNWTT sequence (SEQ ID NO:514) was unexpected due to the presence of Trp (the largest amino acid) in the X +1  position at the center of the required N-X-S/T glycosylation motif. We grafted the GGNWTT sequence (SEQ ID NO:514) as a GlycTag into the internal loop of Im7 at Ala28 28  (Im7-6), and developed an in vitro glycosylation (IVG) method using enzymes and target proteins from CFPS (Methods and  FIG. 18 ). We first validated our method by recapitulating the two-enzyme, native glycosylation system of  A. pleuropneumoniae  on the HMW1ct-WT 47  target protein ( FIG. 18 ). We then assembled IVG reactions containing Im7-6 and NGT synthesized in separate CFPS reactions and a UDP-Glc sugar donor ( FIG. 5 a    and  FIG. 21 ). We purified and analyzed the reaction product with LC-time-of-flight (LC-TOF) mass spectrometry. We found that Im7-6 (containing the GGNWTT GlycTag (SEQ ID NO:514)) was efficiently modified with a single hexose residue ( FIG. 5 a   ). Modification was not observed when NGT was absent ( FIG. 5 a   ) or when the acceptor residue was mutated from Asn to Gln ( FIG. 19 ). 
     We next investigated how the modification efficiencies of peptides in GlycoSCORES screening correlated with modification efficiencies of these sequences in the context of whole proteins. In addition to GGNWTT (SEQ ID NO:514), we investigated 3 additional sequences from GlycoSCORES peptide screens including FANATT, which showed a high glycosylation efficiency (˜75%), YANATS which showed a medium modification efficiency (˜36%) and was used in a previous peptide study of NGT 23 , and NHNETD which showed no detectable modification (data not shown). For comparison to previous studies, we also analyzed a biological consensus sequence for NGT glycosylation (GANATA (SEQ ID NO:515)) derived from an LC-MS/MS study by Naegeli and coworkers in which NGT was expressed in the cytoplasm of  E. coli   20  as well as the optimized PglB GlycTag sequence (DQNATF) 28  (SEQ ID NO:519) which has been used for study of NGT glycosylation at the peptide level 19 . We determined the Michaelis-Menten constants for these sequences along with the GGNWTT sequence (SEQ ID NO:514) used in  FIG. 5 a    using SAMDI ( FIG. 5 b    and data not shown). The relative values of k cat /K m  correlate with GlycoSCORES conversion efficiencies observed in  FIG. 4 c    and  FIG. 17 . For each of the sequences in  FIG. 5 b   , we synthesized a corresponding Im7 variant containing these sequences at the Ala28 internal loop. To minimize the effects of surrounding amino acids and secondary structure on the glycosylation site, we added flexible flanking sequences around all GlycTags inserted into Im7 based on a biological consensus sequence in the form of ATT-X −2 X −1 NX +1 X +2 X +3 -AGG 20 . The average protein expression yields of all Im7 variants was 624±67 μg/mL (data not shown). We performed IVG reactions and analyzed glycosylation using LC-TOF after purification. Modification was quantified by relative peak areas as in previous literature 48  using dominant charge states of the glycosylated and aglycosylated forms of the protein ( FIG. 5 b    and data not shown). Relative peak areas and deconvoluted mass spectra ( FIG. 19 ) of Im7 variants correlated well with the k cat /K M  values of peptide sequences with identical ranking ( FIG. 5 b   ). Of these sequences, GGNWTT (SEQ ID NO:514) showed the highest k cat /K M  value for the peptide substrate and the most efficient modification within the Im7 protein. We also found Im7-6 can be homogeneously glycosylated by increasing the concentration of CFPS-derived NGT in the IVG reaction to 4 μM ( FIG. 19 ). Therefore, we chose GGNWTT (SEQ ID NO:514) as our optimized GlycTag for site-directed protein glycosylation in vivo. 
     Efficient protein glycosylation in the  E. coli  cytoplasm. Next, we investigated the use of the GlycoSCORES-derived GGNWTT GlycTag (SEQ ID NO:514) to direct efficient modification of heterologous proteins in the cytoplasm of living  E. coli  by redesigning the internal protein glycosylation site at Asn297 in human Fc ( FIG. 6 a   ). NGT was co-expressed with Fc variants containing the naturally occurring sequence (QYNSTY (SEQ ID NO:513)), the biological consensus sequence (GANATA) 20  (SEQ ID NO:515), and our GlycoSCORES optimized GlycTag (GGNWTT (SEQ ID NO:514)) in vivo. As with Im7, flexible sequences flanked the site. Using a two-plasmid system in BL21(DE3)  E. coli , we induced expression of the Fc target protein with IPTG then NGT with arabinose. We found that our GlycoSCORES-derived GlycTag (GGNWTT (SEQ ID NO:514)) enabled the most efficient glycosylation, followed by the biological consensus sequence (GANATA) 20  (SEQ ID NO:515), and the naturally occurring sequence (QYNSTY (SEQ ID NO:513)) in Fc ( FIGS. 6 b  and 6 c    and  FIG. 20 ). We used this system to produce homogeneously glycosylated Fc in  E. coli  by extending the co-expression time of the engineered GGNWTT variant with NGT for 4 h ( FIG. 20 ). This same engineered variant of Fc could also be synthesized in CFPS and efficiently glycosylated in vitro ( FIG. 21 ). We completed a similar analysis using a variant of HMW1ct and found that engineered HMW1ct targets also showed trends predicted by GlycoSCORES peptide characterization ( FIG. 17 ) with the optimized GlycTag GGNWTT (SEQ TD NO:514) showing the highest modification followed by GANATA 20  (SEQ ID NO:515) and the naturally occurring NINATS sequence (SEQ ID NO:512) (data not shown). We observed similar expression levels of NGT and Fc or HMW1ct variants across all strains indicating that differences in glycosylation efficiency are due to NGT sequence specificity rather than differences in expression (data not shown). 
     Discussion 
     This paper describes the GlycoSCORES platform, a cell-free approach for rapid determination of GT peptide specificity to improve fundamental understanding of glycosylation systems and guide the efficient glycosylation of diverse proteins in vitro and in vivo. By using more than 3,480 unique peptide substrates and 13,903 unique reaction conditions, GlycoSCORES enabled, to our knowledge, the most complete substrate characterization of any ppGT thus far (data not shown). This dataset further facilitated the selection of efficiently modified NGT peptide substrates ( FIG. 19  and data not shown), for example GGNWTT (SEQ ID NO:514), which was found to direct efficient glycosylation of Im7, HMW1ct, and Fc proteins. Looking forward, our dataset could also be used to make informed, single mutations to improve modification. Future work could also explore how glycosylation of these optimized sequences is affected by protein structure. We further generalized the GlycoSCORES approach to discover two NGT homologs in pathogenic bacteria with conserved peptide specificities, show complex specificity differences between human ppGalNAcTs, and demonstrate a proof of principle for high-throughput analysis of hOGT specificity. These innovations result from the ability to screen substrate residues more completely and determine synergistic residue combinations that are conventionally not tested. 
     When combined with recent advancements in the elaboration of the single glucose residue installed by NGT to human-like glycans using chemoenzymatic transglycosylation techniques 13, 27  and polysialic acids using a fully biosynthetic approach 21 , the deep specificity data and demonstration of highly efficient GlycTags shown in this work may open the door to diverse applications of NGT-based synthetic glycosylation systems just as the design and implementation of OST GlycTags 28  enabled the improvement of biopharmaceuticals and an array of studies using the bacterial OST, PglB, to produce vaccines and therapeutics in  E. coli   8, 9 . NGT systems may complement OST-based methods as they do not require export out of the cytoplasm or lipid-associated substrates 9 . 
     Given the versatility of CFPS for rapid, parallelized expression of diverse enzymes and target proteins and the throughput of SAMDI for rapid detection of glycosylation without radioactively or chemically modified sugars or antibodies, we anticipate the application of GlycoSCORES to a broad range of ppGTs investigations of interest to the glycoengineering community including the further characterization of the ppGalNAcTs, OGTs, and OSTs (which have been recently shown to be produced in CFPS using protein nanodiscs 49 ). GlycoSCORES is also uniquely suited to the engineering of glycosylation enzymes for alternative specificities in vitro, obviating the need for in vivo selection schemes, which have been challenging to develop for glycan modification. Specifically, CFPS reactions can be performed in 96/384 well plates with linear templates, substrate concentrations can be rigorously controlled, and any peptide mass addition can be detected and quantified by SAMDI. An example application is the synthesis and screening of diverse NGT homologs and engineered variants (building off recent work on NGT mutants 13, 24 ) to install GlcNAc onto proteins using a single enzyme or identifying ppGTs that can modify a specific amino acid sequence of interest. 
     In summary, the GlycoSCORES workflow provides a versatile platform for characterizing and engineering GTs. By allowing for detailed characterization of diverse systems in the current and future studies, we expect this platform to enable a deep, quantitative understanding of glycosylation systems and advance compelling biotechnology applications. 
     Methods 
     Solid phase synthesis of peptide arrays. All peptide arrays were synthesized manually using 96-well filter plates (Cat. No. AWFP-F20000, Arctic White LLC) as described previously 31  with some modification. All Fmoc-Amino Acids and Fmoc-Rink Amide MBHA resins were purchased from AnaSpec Inc. All solvents, N,N-dimethylformamide (DMF), dichloromethane (DCM), trifluoroacetic acid (TFA) and piperidine were purchased from Thermo Fisher Scientific. Other chemical reagents used in peptide synthesis were purchased from Sigma-Aldrich unless otherwise noted. Briefly, 10 mg of Fmoc-Rink Amide MBHA resins were placed in each well of 96-well filter plates. Before adding each amino acid, N-terminal fluorenylmethyloxycarbonyl (Fmoc) was deprotected with 300 μL 20% piperidine in DMF, with 600 rpm shaking for 30 min. After 5 washes with DMF, 300 μL 0.1 M Fmoc-Amino Acid, 0.125 M hydroxybenzotriazole (HOBt) and 0.1 M diisopropylcarbodiimide (DIC) were used to add the amino acid onto the resin, with 600 rpm shaking for 2 h. After all amino acids were added onto the resin, Fmoc was deprotected and acetic anhydride (10% in DMF) was used to add an acetyl group on the N-terminal of peptides with 600 rpm shaking for 0.5 h. The resin was washed with DCM 5 times and dried for 1 h, before being cleaved by 500 μL 95% TFA, 2.5% H 2 O, and 2.5% triethylsilane (TES) with 400 rpm shaking for 2 h. After the solvents were evaporated by flowing nitrogen overnight, remaining solids were dissolved with 600 μL H 2 O and transferred to 96-well plates. After lyophilization, the peptides were redissolved in 200 μL 50 mM Tris (pH 8), transferred to 384-well plates (Ref. No. 784201, Greiner Bio One), and stored at −80° C. All peptides had one cysteine to determine the concentration of the peptides and bind to SAMs on gold islands. In most cases, an Arg residue was included N-terminal to the Cys to provide efficient ionization in the mass spectrometry experiments. 
     Preparation of SAMDI plates. 384 SAMDI plates were prepared as previously described 51  with minor modifications. Briefly, 384 islands with 50 nm Ti and 300 nm Au were prepared by evaporation and rinsed in 0.25 mM ethanolic solution containing 60% of a symmetric disulfide presenting tri(ethylene glycol) (EG3 disulfide from ProChimia Surfaces, Poland) and 40% of an asymmetric disulfide presenting one tri(ethylene glycol) and one maleimide (EG3-Maleimide disulfide from ProChimia Surfaces, Poland) at 4° C. for 2 days. The SAMDI plate was ready for use after washing with ethanol, H 2 O, and ethanol then drying with flowing nitrogen. 
     Profiling NGT activity with peptide arrays. A subset of 16-24 peptides were used to measure the average concentration of each peptide library (361 or 380 peptides) using the Ellman test based on manufacture&#39;s protocols (Gold Biotechnology). After reduction with tris(2-carboxyethyl)phosphine (TCEP) reducing gel (Thermo Fisher Scientific), 50 μM peptides were reacted with indicated concentrations of NGT purified by Ni-NTA from living  E. coli  or unpurified NGT produced in  E. coli  CFPS, 2.5 mM UDP-Glc in 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (in HEPES, pH 8) and 500 mM NaCl at 30° C. for indicated times. As a control, the same volume of CFPS after 20 h of sfGFP synthesis was used instead of NGT from CFPS. The reaction was not quenched unless otherwise stated. 2 μL TCEP reducing gel was added to each 10 μL of reaction solutions and incubated at 37° C. for 1 h, before 2 μL reduced solutions were transferred to 384 SAMDI plate using Tecan 96-channel arm and incubated at room temperature for 0.5 h. SAMDI plates were washed with H 2 O, ethanol, H 2 O, and ethanol, and dried with flowing nitrogen. After application of 10 mg/mL of 2′,4′,6′-Trihydroxyacetophenone monohydrate (THAP) matrix (Sigma-Aldrich) in acetone onto the entire SAMDI plate, an Applied Biosystems SciEx MALDI-TOF/TOF 5800 instrument was used to perform mass spectrometry on each spot. Applied Biosystems SciEx Time of Flight Series Explorer Software version 4.1.0 was used to analyze MS spectra. Generally, each 384-well IVG plate was immobilized onto 2 separate 384-well SAMDI plates and analyzed by MALDI separately. The modification efficiency of peptides was calculated using the following equation: 
     
       
         
           
             
               % 
               ⁢ 
               
                   
               
               ⁢ 
               modification 
             
             = 
             
               
                 
                   I 
                   ⁡ 
                   
                     ( 
                     P 
                     ) 
                   
                 
                 
                   RIF 
                   ⁡ 
                   
                     ( 
                     
                       P 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       to 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       S 
                     
                     ) 
                   
                 
               
               
                 
                   
                     I 
                     ⁡ 
                     
                       ( 
                       P 
                       ) 
                     
                   
                   
                     RIF 
                     ⁡ 
                     
                       ( 
                       
                         P 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         to 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         S 
                       
                       ) 
                     
                   
                 
                 + 
                 
                   I 
                   ⁡ 
                   
                     ( 
                     S 
                     ) 
                   
                 
               
             
           
         
       
     
     where I(P) is the intensity of product Glc-peptide in mass spectrometry, I(S) is the intensity of substrate peptide in mass spectrometry, and RIF(P to S) is the relative ionization factor of product to substrate. RIFs equal IF(P)/IF(S) and were determined as described below. Glucose modification efficiencies for peptides are shown as heat maps in  FIGS. 2 and 4  and annotated with numerical values in  FIGS. 9-11  and  FIGS. 15-17  and data not shown. Heat maps for comparison or characterization are arranged by amino acid properties while heat maps for NGT optimization, are ranked in descending order by average modification of all substrates in each row or column and ranked from left to right and top to bottom. As determined by negative controls peptide arrays using CFPS synthesizing sfGFP rather than NGT (data not shown), glucose modification efficiencies of less than 0.03 were regarded as background. NGT homologs MhNGT and HdNGT were produced in  E. coli  CFPS and profiled with peptide arrays the same way as NGT ( FIG. 12  and data not shown). 
     Measuring relative ionization factors. Relative ionization factors (RIFs) were determined by measurements of mass spectra intensity/concentration for glycosylated and aglycosylated samples. After reduction with TCEP reducing gel, 50 μM peptides were reacted with 2.5 mM UDP-Glc and 10 μM purified NGT or 0.575 μM CFPS NGT in 100 mM HEPES (pH 8) and 500 mM NaCl, at 30° C. for 4 h to achieve more than 70% glucose modification (glycosylated samples). Identical reactions without UDP-Glc were used as control reactions to provide the same total concentration of peptides (aglycosylated samples). The reactions were quenched by placing the reaction plates at 60° C. for 20 min. Glycosylated and aglycosylated samples were mixed at a 1:1 ratio and reduced with TCEP reducing gel, and mass spectra for glycosylated, aglycosylated and mixed samples were collected by SAMDI. The aglycosylated samples always showed no detectable glucose modification. Relative ionization factors were calculated using the equation below. 
     
       
         
           
             RIF 
             = 
             
               
                 % 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 
                   I 
                   ⁡ 
                   
                     ( 
                     Re 
                     ) 
                   
                 
                 * 
                 % 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 
                   I 
                   ⁡ 
                   
                     ( 
                     Mix 
                     ) 
                   
                 
               
               
                 
                   % 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   
                     I 
                     ⁡ 
                     
                       ( 
                       Re 
                       ) 
                     
                   
                   * 
                   % 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   
                     I 
                     ⁡ 
                     
                       ( 
                       Mix 
                       ) 
                     
                   
                 
                 + 
                 
                   % 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   
                     I 
                     ⁡ 
                     
                       ( 
                       Re 
                       ) 
                     
                   
                 
                 - 
                 
                   2 
                   * 
                   % 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   
                     I 
                     ⁡ 
                     
                       ( 
                       Mix 
                       ) 
                     
                   
                 
               
             
           
         
       
     
     Where % I(Re) is the intensity of the glycosylated product peptide (I(P g )) divided by the sum of the intensities of the glycosylated product peptide (I(P g )) and aglycosylated substrate peptide (I(S g )) in the glycosylated samples or I(P g )/(I(P g )+I(S g )). % I(Mix) is the intensity of the glycosylated product peptide (I(P m )) divided by the sum of the intensities of the glycosylated product peptide (I(P m )) and aglycosylated substrate peptide(I(S m )) in a 1:1 ratio mixture of glycosylated and aglycosylated samples or I(P m )/(I(P m )+I(S m )). A subset of 20-24 peptides were used to measure the relative ionization factor (RIF) of each peptide library. The RIFs of peptides for which reaction kinetics data was collected were also determined (data not shown). 
     Determining sugar donor specificity of NGT. Six peptides were used to profile the monosaccharide selectivity of NGT. 50 μM peptides were reacted with 0.1-0.2 μM purified NGT, 1 mM UDP-Glc, UDP-Gal, GDP-Man, UDP-GlcNAc, UDP-GalNAc or UDP-Xyl in 100 mM HEPES (pH 8) and 500 mM NaCl at 30° C. for 1 h, 4 h or 21 h. After reduction with TCEP reducing gel, the percentage intensity of Glc-peptide was recorded by SAMDI. For testing of sugar donor selectivity of glucose, galactose, or xylose modification with the X −1 NX +1 TRC peptide library, 1 mM UDP-Glc, UDP-Gal or UDP-Xyl and indicated concentration of purified NGT were used. UDP-Xyl was purchased from Carbosource Services. Other sugar donors were purchased from Sigma-Aldrich. 
     Measuring reaction kinetics parameters of selected GlycTag peptides. Various (6-8) concentrations of selected HPLC-purified peptides were reacted with indicated concentrations of NGT and 10 mM UDP-Glc in 100 mM HEPES (pH 8) and 500 mM NaCl at 30° C. for a series of reaction times (15 min to 2 h). Reactions were quenched using 2 μL of 2 M HCl per 10 μL of reaction solution. After neutralization with 2 μL 2 M K 2 CO 3  and reduction with TCEP reducing gel, the modification efficiency was determined by SAMDI. Initial reaction velocities were calculated using the slopes in the linear time-frame of each initial peptide concentration. K M  and k cat  and associated errors were then determined by non-linear fitting to the Michaelis-Menten formula using OriginPro 9 software. 
     Using GlycoSCORES to screen peptide selectivity of human O-linked GTs produced in CFPS. To demonstrate the applicability of GlycoSCORES to the study mammalian O-linked GTs, ppGalNAcT1, ppGalNAcT2, and hOGT were produced in  E. coli  CFPS. While hOGT was synthesized in CFPS the same way as NGT, the ppGalNAcTs were synthesized in CFPS under oxidizing conditions to allow for formation of disulfide bonds. Oxidizing conditions were achieved using standard CFPS reactions were modified as described previously 52 , supplemented with 14.3 μM iodoacetamide, 1 mM glutathione, 4 mM glutathione disulfide, and 3.16 μM  E. coli  disulfide bond isomerase (DsbC). For GlycoSCORES screening of ppGalNAcTs, 100 μM of each peptide from peptide array AX −1 TX +I APRC was reacted with 0.024 μM CFPS ppGalNAcT1 or 0.04 μM ppGalNAcT2, 1 mM UDP-GalNAc in 100 mM HEPES (pH 7.5), and 3 mM Mn 2+  at 37° C. for 1 h followed by quenching with 5 mM EDTA. As a control, the same volume of CFPS after 20 h of sfGFP synthesis was used instead of ppGalNAcTs from CFPS. GlycoSCORES screening of hOGT was completed similarly, with 50 μM of each peptide variant of the sequence PPVSRC reacted with 0.62 μM hOGT made in CFPS and 2.5 mM UDP-G1cNAc in 20 mM Tris (pH 7.4), 125 mM NaCl, and 1 mM EDTA for 21 h at 37° C. After reduction with TCEP reducing gel and maleimide capture, the relative percentage intensities of the GalNAc or GlcNAc-modified and unmodified peptides were recorded by SAMDI as described for NGT. 
     Plasmid construction and molecular cloning. Plasmids used in this study with sources and details are reported in “Design of glycosylation sites by rapid expression and high-throughput characterization of N-glycosyltransferase,” by Weston Kightlinger, Liang Lin, Madisen Rosztoczy, Matthew P. DeLisa, Milan Mrksich, and Michael C. Jewett, Nat. Chem. Biol., 2018 May 7, doi: 10.1038/s41589-018-0051-2, which content is incorporated herein by reference in its entirety. The wildtype Im7 coding sequence (Uniprot: IMM7_ECOLX) was PCR amplified from pBR322.Im7 and assembled into the pJL1 CFPS vector between the NdeI and SalI sites using Gibson Assembly to produce pJL1.Im7-0s. Wildtype IgG1 constant Fc region (Δ1-98 Uniprot: IGHG1_HUMAN) was synthesized by Twist Bioscience and assembled into a variant of pET22b with redesigned restriction sites (pETBCS.NS) using restriction ligation to form pETBCS.NS.Fc-0s. The wildtype sequence for HMW1ct (Δ1-1203 GenBank: AD096128.1) was synthesized by Life Technologies and assembled into pJL1 between NdeI and SalI sites to form pJL1.HMW1ct-WT. A variant sequence of HMW1ct with N/Q substitutions at all naturally occurring N-X-S/T sites except at N1366 was synthesized by Life Technologies and assembled into pET.BCS.NS to form pET.BCS.NS.HMW1ct-0 using restriction and ligation at NdeI and SalI sites. Variants of the N26_T31 NVAAT loop in Im7-0s, the Q178_Y183 QYNSTY (SEQ ID NO:513) naturally occurring glycosylation sequence in Fc-0s, and the N1364_S1370 naturally occurring glycosylation sequence in HMW1ct-0 were constructed by inverse PCR with 18 bp of overlapping 5′ homology and recircularized by one-piece Gibson Assembly. All variants of Im7, Fc, and HMW1ct contained C-terminal 6×His-tags. Wildtype NGT sequence (Uniprot: NGT_ACTP2) was synthesized by Twist Bioscience and assembled into pJL1 between NdeI and SalI sites, pET.21b between NcoI and XhoI sites with a C-terminal 6×His-tag, and pMAF10 between NcoI and HindIII with a C-terminal 1×FLAG tag by Gibson Assembly. The α-1,6 glucose polymerase from  A. pleuropneumoniae  (AGT, Uniprot: GTF_ACTP7) was ordered from Twist Bioscience in pJL1 with a customized ribosome binding site designed for maximum translation initiation rate using the RBS Calculator v2.0 53 . Codon optimized sequences for MhNGT (Uniprot: A0A0B5BRN9_MANHA) and HdNGT (Uniprot: Q7VKK3_HAEDU) were ordered from Integrated DNA Technologies with C-terminal Strep tags and placed into PJL1 using Gibson assembly. Human ppGalNAcT1 (CGAT1_HUMAN) and ppGalNAcT2 (CGAT2_HUMAN) truncated without the N-terminal 40 aa (Δ40) were also cloned into PJL1 using Gibson Assembly either with or without N-terminal CAT-Strep-Linker fusions. The coding sequence for hOGT (OGT1_HUMANΔ1-313) was PCR amplified from pET42a.hOGT 46  and cloned into PJL1 using Gibson Assembly. 
     Preparation of cell extracts for CFPS. Crude extracts for CFPS were generated from a genomically recoded release factor 1 (RF1) deficient  E. coli  strain ( E. coli  C321.ΔA.759), based on  E. coli  C321.ΔA 54 . Cell growth, harvest, and lysis were performed as described in Kwon and Jewett 55 . Briefly,  E. coli  cells were grown in 1 L of 2×YTPG (yeast extract 10 g/L, tryptone 16 g/L, NaCl 5 g/L, K 2 HPO 4  7 g/L, KH 2 PO 4  3 g/L, and glucose 18 g/L, pH 7.2) in a 2.5 L Tunair flask at 34° C. and 250 rpm with initial inoculation to OD600=0.08. At OD600=3.0, cells were pelleted by centrifugation at 5,000×g at 4° C. for 15 min. The pellets were washed three times with cold S30 buffer (10 mM Tris-acetate pH 8.2, 14 mM magnesium acetate, 60 mM potassium acetate, 2 mM dithiothreitol (DTT)) and flash frozen on liquid nitrogen and stored at −80° C. Cells were thawed, resuspended in 0.8 mL of S30 buffer per gram wet weight, and lysed in 1.4 mL aliquots on ice using a Q125 Sonicator (Qsonica) for three pulses (50% amplitude, 45 s on and 59 s off). After sonication, 4 μL of DTT (1 M) was added followed by centrifugation at 12,000×g and 4° C. for 10 min. The supernatant was incubated at 37° C. at 250 rpm for 1 h for a run-off reaction and centrifuged again at 10,000×g at 4° C. for 10 min. The supernatant was flash-frozen on liquid nitrogen and stored at −80° C. until use. 
     Cell-free protein synthesis. CFPS reactions were conducted using a PANOx-SP crude lysate system 56 . A standard reaction contained 1.2 mM ATP; 0.85 mM each of GTP, UTP, and CTP; 34 μg/mL folinic acid; 170 μg/mL of  E. coli  tRNA mixture; 16 μg/mL purified T7 RNA polymerase; 2 mM for each of the 20 standard amino acids; 0.33 mM nicotinamide adenine dinucleotide (NAD); 0.27 mM coenzyme-A (CoA); 1.5 mM spermidine; 1 mM putrescine; 4 mM sodium oxalate; 130 mM potassium glutamate; 10 mM ammonium glutamate; 12 mM magnesium glutamate; 57 mM HEPES, pH 7.2; 33 mM phosphoenolpyruvate (PEP); 13.3 μg/mL plasmid template of interest; and 27% v/v of  E. coli  cell extract.  E. coli  total tRNA mixture (from strain MRE600) and phosphoenolpyruvate was purchased from Roche Applied Science. ATP, GTP, CTP, UTP, 20 amino acids and other materials were purchased from Sigma-Aldrich. Plasmid DNA for cell-free was purified from DH5-α  E. coli  strain (NEB) using ZymoPURE Midi Kit (Zymo Research). NGT and AGT were synthesized in 50 μL batch reactions in 2.0 mL microtubes and Im7 and HMW1ct-WT target proteins were synthesized in 15 μL batch reactions in 1.5 mL microtubes. The CFPS reactions were carried out at 20° C. for 20 h. 
     Quantification of CFPS yields. Total and soluble CFPS yields were quantified using CFPS reactions identical to those used for NGT, AGT, Im7, HdNGT, MhNGT, ppGalNAcT1, ppGalNAcT2, and hOGT synthesis supplemented with 10 μM  14 C-leucine (Perkin-Elmer). Protein quantification for triplicate CFPS reactions was completed using trichloroacetic acid (TCA) protein precipitation followed by radioactivity quantification using a Microbeta2 liquid scintillation counter (Perkin Elmer) according to established protocols 57 . Soluble fractions were taken after centrifugation at 12,000×g for 15 min at 4° C. CFPS yields of sfGFP were quantified as described previously 55  using a multi-well fluorimeter (Synergy2, BioTek) and converted to μg/mL yields using a previously determined standard curve based on  14 C leucine incorporation assays 58 . 
     Autoradiograms of CFPS proteins. After synthesis in  14 C-leucine supplemented CFPS reactions, 2 μL of each sample was loaded onto a 4-12% Bolt Bis-Tris Plus SDS-PAGE gels (Invitrogen) and run in MOPS buffer at 150 V for 70 min. The gels were stained using InstantBlue (Expedeon) and destained in water. The gels were incubated in gel drying solution (Bio-rad), dried overnight between cellophane films in a GelAir Dryer (Bio-Rad) without heating, and exposed for 48 h on a Storage Phosphor Screen (GE Healthcare). Autoradiogram images were acquired using Typhoon FLA7000 imager (GE Healthcare). The same coomassie stained gels were imaged using a GelDoc XR+ Imager for molecular weight standard references (Bio-rad). 
     Production and purification of NGT from  E. coli . NGT was purified as described previously 23  with minor modifications. Briefly, BL21 (DE3) cells were transformed with pET21b.NGT plasmid by electroporation. An overnight culture was inoculated in carbenicillin (CARB) LB media. Fresh CARB LB was inoculated at initial OD600=0.08 and the cells were grown at 37° C. at 250 rpm to 0.6-0.8 OD and induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h at 30° C. The cells were pelleted by centrifugation at 8,000×g for 10 min at 4° C., resuspended in Buffer 3 (20 mM Tris-HCl and 250 mM NaCl, pH 8.0), pelleted again by centrifugation at 8,000×g for 10 min at 4° C. and frozen at −80° C. The pellets were then thawed and resuspended in 5 mL Buffer 3 with 20 mM Imidazole per gram wet pellet weight; supplemented with 70 μL of 10 mg/mL lysozyme (Sigma), 1 μL Benzonase (Millipore), and 1× Halt protease inhibitor (Thermo Fisher Scientific); lysed by single pass homogenization at 21,000 psig (Avestin); and centrifuged at 15,000×g for 20 min at 4° C. The supernatant was applied to an Ni-NTA agarose column (Qiagen) equilibrated with Buffer 3 with 20 mM imidazole, washed with 10 column volumes of Buffer 3 with 40 mM imidazole, and eluted with 4 column volumes of Buffer 3 with 500 mM Imidazole. The elution was dialyzed against 50 mM HEPES 200 mM NaCl, pH 7.0, supplemented with 5% glycerol, and flash frozen at −80° C. NGT concentration was quantified using Image Lab software version 6.0.0 densitometry with BSA standard curve after separation by SDS-PAGE, staining with InstantBlue coomassie stain, and destaining in water. 
     In vitro glycosylation of protein substrates. IVG reactions were assembled in standard 0.2 mL tubes from completed CFPS reactions containing targets and enzymes at concentrations determined by  14 C-leucine incorporation. Im7 glycosylation reactions contained 5 μM of one Im7 variant, 0.1 μM NGT, and 2.5 mM UDP-Glc in the final reaction. Each reaction contained a total of 5 μL UDP-Glc and 25 μL CFPS reaction (remaining CFPS reaction volume up to 25 μL was filled by a completed CFPS reaction which synthesized sfGFP). Similarly, HMW1ct-WT IVG reactions contained 5 μM HMW1ct-WT, 0.1 μM NGT, and 2 μM AGT and 2.5 mM UDP-Glc in the final reaction. Each IVG reaction contained 10 μL completed CFPS reaction and 2 μL UDP-Glc. IVG reactions for Im7 and HMW1ct-WT were performed at 30° C. for 2.5 h and 16 h, respectively. 
     Western blotting of HMW1ct-WT. Completed HMW1ct-WT IVG reactions (1 μL) were loaded onto a 4-12% Bolt Bis-Tris SDS-PAGE gel in MOPS buffer and run at 130 V for 100 min. The gel was then transferred onto a 0.2 μM PVDF membrane (Bio-Rad) using the Trans-Blot SD semi-dry blotting system (Bio-Rad) using 80% MOPS and 20% Methanol buffer. The target protein was detected by blocking the membrane in 5% milk in PBS with 0.1% Tween 20 and then incubating with a polyclonal His antibody (Abcam, ab1187) diluted 1:7,500 in PBS with 1% milk for 45 min. The poly-α-Glucose moiety installed by NGT and AGT was detected using a ConA lectin blot using an identical gel with identical membrane and transfer conditions. The ConA blot was blocked with Carbo-free solution (Vector Laboratories) for 1 h and probed with 5 μg/mL ConA-HRP (Sigma, L6397-1MG) diluted in Carbo-free solution supplemented with 1 mM MgCl 2 , 1 mM MnCl 2 , 0.1% Tween, and 1 mM CaCl 2  for 1 h. Blots were imaged using Western-Sure Chemiluminescent substrate on an Odessey Fc (Li-Cor) imager. 
     Purification from in vitro glycosylation reactions. Purification of Im7 from IVG reactions was completed using Dyna-His tag beads (Thermo Fisher Scientific). The 30 μL IVGs were diluted to 120 μL in Buffer 1 (50 mM NaH 2 PO 4  and 300 mM NaCl, pH 8.0) with a final concentration of 10 mM imidazole and incubated at room temperature for 5 min on a roller with 20 μL of beads. The beads were then washed with 120 μL of 20 mM imidazole in Buffer 1 four times using a 96 well plate magnetic tube rack (Life Technologies) for separations. The samples were then eluted using 30 μL of 500 mM imidazole in Buffer 1. The samples were dialyzed against Buffer 2 (20 mM NaH 2 PO 4  and 150 mM NaCl, pH 7.5) in 3.5 kDa MWCO 96-well plate dialysis cassettes (Thermo Fisher Scientific). After dialysis, 10 μL was injected into LC-TOF for analysis. 
     Production of glycosylated proteins in cells.  E. coli  BL21 (DE3) cells were transformed first with pMAF10.NGT by electroporation and selected on trimethoprim (TMP) LB agar plates. A colony was picked and prepared for calcium-chloride transformation and transformed with pETBCS.NS vectors containing Fc or HMW1ct target proteins and selected on TMP+CARB LB agar plates. Colonies were grown to mid-exponential phase and glycerol stocked. The glycerol stocks were used to inoculate overnight cultures in TMP+CARB LB media. Fresh cultures in TMP+CARB were inoculated at initial OD600=0.08 and grown at 37° C. at 250 rpm. For HMW1ct sequence variants, the target protein was induced at 0.6-0.8 OD for 1 h with 400 μM IPTG at 30° C. followed by NGT induction with 0.2% arabinose for 2 h at 30° C. For Fc sequence variants, the target protein was induced for 2 h followed by NGT induction for 30 min (unless otherwise noted) at identical inducer concentrations. The cells were then pelleted by centrifugation at 4° C. for 2 min at 10,000×g, resuspended in Buffer 1, centrifuged at 4° C. for 2 min at 10,000×g, frozen on liquid nitrogen, and stored at −80° C. The pellets were thawed and resuspended in 630 μL of Buffer 1 with 10 mM imidazole and supplemented with 70 μL of 10 mg/mL lysozyme (Sigma), 1 μL Benzonase (Millipore), and 1× Halt protease inhibitor (Thermo Fisher Scientific). After 15 min of thawing and resuspension, the cells were incubated for 15 min on ice and sonicated for 45 s at 50% amplitude and then spun at 12,000×g for 15 min. The supernatant was then loaded onto Ni-NTA His-tag spin columns (Qiagen) pre-equilibrated with 10 mM imidazole in Buffer 1. The columns were washed 3 times with 30 mM imidazole and eluted with 2×100 μL 500 mM imidazole. Samples were then dialyzed with 10 kDa MWCO MINI slide-α-lyzers (Thermo Fisher Scientific) overnight. Protein concentrations were quantified using Image Lab software densitometry with BSA ladder standard after separation by SDS-PAGE, 1 h stain with InstantBlue, and 1 h destain in water. Prior to injection into LC-TOF, purified Fc was incubated with 50 mM DTT for 1 h at room temperature to reduce disulfide linkages. 
     LC-TOF Analysis of Glycoprotein Modification. Purified proteins from CFPS of Im7 or in vivo expression of Fc and HMW1ct were injected onto an Agilent 1200 HPLC equipped with an XBridge BEH300 Å C4 3.5 μm 2.1 mm×50 mm reverse-phase column (186004498 Waters Corporation) with a 10 mm guard column of identical packing (186007230 Waters Corporation) coupled to an Agilent 6210A ESI-TOF mass spectrometer. The chromatographic separation method was based on manufacturer instructions for XBridge column with minor modifications. Solvent A was 95% H 2 O and 5% acetonitrile (ACN) with 0.1% formic acid and solvent B was 100% ACN with 0.1% formic acid. The separation was completed at a flow rate of 0.4 mL/min with a column temperature of 50° C. Solvent conditions were held at 15.8% B for 1 min, then the target protein of interest was eluted during a 12 min gradient from 15.8% to 65.8% B. The column was then washed and re-equilibrated using a 2 min gradient from 65.8-69.9% B, a 2 min hold at 100% B, and a 6 min hold at 15.8% B. Purified Fc after in vitro synthesis and glycosylation was injected into a Bruker Elute UPLC system, separated using the same chromatography methods as listed above, and analyzed by an Impact-II UHR TOF-MS system (Bruker Daltonics, Inc.). External calibration was completed prior to analysis of all proteins. 
     LC-TOF Data Analysis. Data from Agilent 6210A was processed using Agilent Mass Hunter software version B.04.00. Methods for quantification of relative peak areas for glycosylated and aglycosylated glycoforms were adapted from previous works 48, 59 . Extracted ion chromatograms (EICs) were created using theoretical values for the most dominant charge states from the glycosylated and aglycosylated samples ±0.5 Da. Protonated charge states +12 to +14, +29 to +36, and +34 to +43 were used to quantify the relative peak areas for Im7, Fc, and HMW1ct, respectively. EIC peaks corresponding with retention times of each protein (data not shown) were then integrated and used for quantification of relative peak areas, defined as Glc 1 /(Glc 0 +Glc 1 ). Deconvoluted spectra were produced using Agilent Mass Hunter maximum entropy deconvolution using MS peaks within m/z range 700-2000 into mass ranges of 10,000-15,000 u; 25,000-30,000 u; and 32,500-37,500 u for Im7, Fc, and HMW1ct, respectively. Isotope widths were calculated by Mass Hunter for deconvolution mass ranges at 7.1, 10.5, and 11.6 u for Im7, Fc, and HMW1ct, respectively. Data from Impact-II UHR TOF-MS was performed using Bruker Compass Hystar software version 4.1. Deconvolution was performed using maximum entropy deconvolution using MS peaks within m/z range 700-2000 into a mass range of 20,000-30,000 u. Raw data was then plotted and annotated using R Studio. Deconvolutions used full mass spectra averaged across the entire peak width of the proteins of interest (encompassing the full elution of the glycosylated and aglycosylated glycoforms). Deconvoluted masses and errors compared to calculated values also were generated and analyzed (data not shown). 
     Statistical Analysis. Two-tailed Student&#39;s t-tests and resulting p-values were calculated in Microsoft Excel 2016 assuming unequal variances and two-tailed distributions to assign significance to observed differences in relative peak areas for GlycTag variants of Im7, HMW1, and Fc. In these cases, n=3 independent IVG reactions were performed for analysis of Im7 while n=3 independent  E. coli  expression cultures were completed for analysis of HMW1 and Fc. 
     Example 3—Modification Efficiencies 
     Using the methods disclosure herein, the efficiency of modification of various peptide sequences comprising 4-mers by different prokaryotic N-glycosyltransferases was tested. The results are presented in Tables 1-5 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Modification Efficiencies for 4-mer Peptides 
               
               
                 by the N-glycosyltransferase from  
               
               
                 
                   Actinobacillus pleuropneumoniae 
                 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID 
                   
                 MODIFICATION 
               
               
                 NO 
                 SEQUENCE 
                 EFFICIENCY 
               
               
                   
               
               
                 1 
                 ANVT 
                 0.984415739 
               
               
                   
               
               
                 2 
                 PNVT 
                 0.974081648 
               
               
                   
               
               
                 3 
                 SNVS 
                 0.970991647 
               
               
                   
               
               
                 4 
                 ANVS 
                 0.961116293 
               
               
                   
               
               
                 5 
                 WNVT 
                 0.959477963 
               
               
                   
               
               
                 6 
                 ANIT 
                 0.949681393 
               
               
                   
               
               
                 7 
                 SNVT 
                 0.948773939 
               
               
                   
               
               
                 8 
                 GNWT 
                 0.940868639 
               
               
                   
               
               
                 9 
                 PNVS 
                 0.937421199 
               
               
                   
               
               
                 10 
                 PNIT 
                 0.935753551 
               
               
                   
               
               
                 11 
                 HNVT 
                 0.92947334 
               
               
                   
               
               
                 12 
                 WNIT 
                 0.927501013 
               
               
                   
               
               
                 13 
                 ANHT 
                 0.92009282 
               
               
                   
               
               
                 11 
                 HNVS 
                 0.918469123 
               
               
                   
               
               
                 15 
                 NNVT 
                 0.912108093 
               
               
                   
               
               
                 16 
                 MNVS 
                 0.911819033 
               
               
                   
               
               
                 17 
                 ANAT 
                 0.910075484 
               
               
                   
               
               
                 18 
                 ANYT 
                 0.90999696 
               
               
                   
               
               
                 19 
                 TNVT 
                 0.909628783 
               
               
                   
               
               
                 20 
                 SNHT 
                 0.905957216 
               
               
                   
               
               
                 21 
                 PNYT 
                 0.89630672 
               
               
                   
               
               
                 22 
                 PNHT 
                 0.893750169 
               
               
                   
               
               
                 23 
                 PNAT 
                 0.89193703 
               
               
                   
               
               
                 24 
                 WNVS 
                 0.891025101 
               
               
                   
               
               
                 25 
                 ENVT 
                 0.890314969 
               
               
                   
               
               
                 26 
                 GNVT 
                 0.884287478 
               
               
                   
               
               
                 27 
                 PNIS 
                 0.882098631 
               
               
                   
               
               
                 28 
                 TNVS 
                 0.869468635 
               
               
                   
               
               
                 29 
                 ANIS 
                 0.86938912 
               
               
                   
               
               
                 30 
                 PNRT 
                 0.866193568 
               
               
                   
               
               
                 31 
                 ANFT 
                 0.863314885 
               
               
                   
               
               
                 32 
                 NNHT 
                 0.86237279 
               
               
                   
               
               
                 33 
                 WNAT 
                 0.85991535 
               
               
                   
               
               
                 34 
                 MNHT 
                 0.854975457 
               
               
                   
               
               
                 35 
                 PNTT 
                 0.853226526 
               
               
                   
               
               
                 36 
                 TNHT 
                 0.844442897 
               
               
                   
               
               
                 37 
                 HNHT 
                 0.842703071 
               
               
                   
               
               
                 38 
                 HNIT 
                 0.841062558 
               
               
                   
               
               
                 39 
                 PNFT 
                 0.840189685 
               
               
                   
               
               
                 40 
                 PNMT 
                 0.839913026 
               
               
                   
               
               
                 41 
                 SNAT 
                 0.838341214 
               
               
                   
               
               
                 42 
                 MNVT 
                 0.837446553 
               
               
                   
               
               
                 43 
                 WNHT 
                 0.83348684 
               
               
                   
               
               
                 44 
                 PNST 
                 0.827083781 
               
               
                   
               
               
                 45 
                 PNLT 
                 0.82541612 
               
               
                   
               
               
                 46 
                 ANRT 
                 0.820621518 
               
               
                   
               
               
                 47 
                 VNVT 
                 0.808053372 
               
               
                   
               
               
                 48 
                 MNAT 
                 0.790260086 
               
               
                   
               
               
                 49 
                 ANTT 
                 0.787236781 
               
               
                   
               
               
                 50 
                 ENAT 
                 0.787179032 
               
               
                   
               
               
                 51 
                 GNHT 
                 0.785556357 
               
               
                   
               
               
                 52 
                 ANST 
                 0.784629132 
               
               
                   
               
               
                 53 
                 TNAT 
                 0.776112522 
               
               
                   
               
               
                 54 
                 NNRT 
                 0.767139721 
               
               
                   
               
               
                 55 
                 NNAT 
                 0.766385598 
               
               
                   
               
               
                 56 
                 ENHT 
                 0.76559028 
               
               
                   
               
               
                 57 
                 ANMT 
                 0.762942447 
               
               
                   
               
               
                 58 
                 WNIS 
                 0.746004631 
               
               
                   
               
               
                 59 
                 SNIT 
                 0.745271788 
               
               
                   
               
               
                 60 
                 HNAT 
                 0.742714018 
               
               
                   
               
               
                 61 
                 MNIT 
                 0.73927687 
               
               
                   
               
               
                 62 
                 DNHT 
                 0.728678043 
               
               
                   
               
               
                 63 
                 ENRT 
                 0.727085969 
               
               
                   
               
               
                 64 
                 GNWS 
                 0.720023034 
               
               
                   
               
               
                 65 
                 NNVS 
                 0.714661402 
               
               
                   
               
               
                 66 
                 HNIS 
                 0.70396499 
               
               
                   
               
               
                 67 
                 PNHS 
                 0.698764486 
               
               
                   
               
               
                 68 
                 VNAT 
                 0.698207647 
               
               
                   
               
               
                 69 
                 ANYS 
                 0.697516068 
               
               
                   
               
               
                 70 
                 HNRT 
                 0.696238004 
               
               
                   
               
               
                 71 
                 ANLT 
                 0.695723705 
               
               
                   
               
               
                 72 
                 WNRT 
                 0.693312013 
               
               
                   
               
               
                 73 
                 ANHS 
                 0.691828284 
               
               
                   
               
               
                 74 
                 INVT 
                 0.689347628 
               
               
                   
               
               
                 75 
                 VNIT 
                 0.687272402 
               
               
                   
               
               
                 76 
                 NNHS 
                 0.684843747 
               
               
                   
               
               
                 77 
                 SNRT 
                 0.682056263 
               
               
                   
               
               
                 78 
                 MNHS 
                 0.681569621 
               
               
                   
               
               
                 79 
                 PNYS 
                 0.676235848 
               
               
                   
               
               
                 80 
                 HNHS 
                 0.674735108 
               
               
                   
               
               
                 81 
                 MNRT 
                 0.672379595 
               
               
                   
               
               
                 82 
                 LNVT 
                 0.672374083 
               
               
                   
               
               
                 83 
                 ENVS 
                 0.658378162 
               
               
                   
               
               
                 84 
                 TNST 
                 0.642444026 
               
               
                   
               
               
                 85 
                 PNMS 
                 0.635956603 
               
               
                   
               
               
                 86 
                 SNYT 
                 0.617741657 
               
               
                   
               
               
                 87 
                 GNVS 
                 0.611666564 
               
               
                   
               
               
                 88 
                 ENST 
                 0.604165847 
               
               
                   
               
               
                 89 
                 SNHS 
                 0.59371954 
               
               
                   
               
               
                 90 
                 MNMT 
                 0.554727063 
               
               
                   
               
               
                 91 
                 MNTT 
                 0.550210797 
               
               
                   
               
               
                 92 
                 SNTT 
                 0.549557128 
               
               
                   
               
               
                 93 
                 ANMS 
                 0.533618588 
               
               
                   
               
               
                 94 
                 LNIT 
                 0.528844233 
               
               
                   
               
               
                 95 
                 MNIS 
                 0.528587676 
               
               
                   
               
               
                 96 
                 GNYT 
                 0.519494028 
               
               
                   
               
               
                 97 
                 SNST 
                 0.515592117 
               
               
                   
               
               
                 98 
                 TNHS 
                 0.509141636 
               
               
                   
               
               
                 99 
                 DNVT 
                 0.507296409 
               
               
                   
               
               
                 100 
                 QNVT 
                 0.505578004 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 BIN 
                 FREQUENCY 
               
               
                   
               
               
                   
                 0 
                 9 
               
               
                   
               
               
                   
                 0.05 
                 395 
               
               
                   
               
               
                   
                 0.1 
                 55 
               
               
                   
               
               
                   
                 0.15 
                 37 
               
               
                   
               
               
                   
                 0.2 
                 24 
               
               
                   
               
               
                   
                 0.25 
                 19 
               
               
                   
               
               
                   
                 0.3 
                 20 
               
               
                   
               
               
                   
                 0.35 
                 15 
               
               
                   
               
               
                   
                 0.4 
                 13 
               
               
                   
               
               
                   
                 0.45 
                 16 
               
               
                   
               
               
                   
                 0.5 
                 13 
               
               
                   
               
               
                   
                 0.55 
                 15 
               
               
                   
               
               
                   
                 0.6 
                 3 
               
               
                   
               
               
                   
                 0.65 
                 5 
               
               
                   
               
               
                   
                 0.7 
                 17 
               
               
                   
               
               
                   
                 0.75 
                 9 
               
               
                   
               
               
                   
                 0.8 
                 10 
               
               
                   
               
               
                   
                 0.85 
                 12 
               
               
                   
               
               
                   
                 0.9 
                 15 
               
               
                   
               
               
                   
                 0.95 
                 15 
               
               
                   
               
               
                   
                 1 
                 5 
               
               
                   
               
               
                   
                 More 
                 0 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Modification Efficiencies for 4-mer Peptides 
               
               
                 by the N-glycosyltransferase from 
               
               
                 
                   Escherichia coli 
                 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID 
                   
                 MODIFICATION 
               
               
                 NO 
                 SEQUENCE 
                 EFFICIENCY 
               
               
                   
               
               
                 101 
                 VNIS 
                 0.82796747 
               
               
                   
               
               
                 102 
                 PNMS 
                 0.753577002 
               
               
                   
               
               
                 103 
                 PNYS 
                 0.735004331 
               
               
                   
               
               
                 104 
                 LNIS 
                 0.731581868 
               
               
                   
               
               
                 105 
                 PNFS 
                 0.698269083 
               
               
                   
               
               
                 106 
                 PNIS 
                 0.668808087 
               
               
                   
               
               
                 107 
                 PNLS 
                 0.627359189 
               
               
                   
               
               
                 108 
                 PNIT 
                 0.60279085 
               
               
                   
               
               
                 109 
                 SNIS 
                 0.59009408 
               
               
                   
               
               
                 110 
                 WNGS 
                 0.584266436 
               
               
                   
               
               
                 111 
                 GNIS 
                 0.566026277 
               
               
                   
               
               
                 112 
                 ANVS 
                 0.564339393 
               
               
                   
               
               
                 113 
                 GNVS 
                 0.561344135 
               
               
                   
               
               
                 114 
                 LNVS 
                 0.550034231 
               
               
                   
               
               
                 115 
                 PNVS 
                 0.544920711 
               
               
                   
               
               
                 116 
                 INVS 
                 0.541576458 
               
               
                   
               
               
                 117 
                 TNIS 
                 0.532404343 
               
               
                   
               
               
                 118 
                 YNAT 
                 0.528232857 
               
               
                   
               
               
                 119 
                 LNHS 
                 0.528190841 
               
               
                   
               
               
                 120 
                 ANIT 
                 0.523701957 
               
               
                   
               
               
                 121 
                 SNVS 
                 0.519382814 
               
               
                   
               
               
                 122 
                 VNMS 
                 0.51810446 
               
               
                   
               
               
                 123 
                 PNYT 
                 0.517274136 
               
               
                   
               
               
                 124 
                 VNVS 
                 0.516905557 
               
               
                   
               
               
                 125 
                 PNGS 
                 0.512088764 
               
               
                   
               
               
                 126 
                 PNFT 
                 0.507277631 
               
               
                   
               
               
                 127 
                 MNHS 
                 0.492958628 
               
               
                   
               
               
                 128 
                 ANIS 
                 0.49169052 
               
               
                   
               
               
                 129 
                 MNIS 
                 0.477399865 
               
               
                   
               
               
                 130 
                 WNGT 
                 0.475704948 
               
               
                   
               
               
                 131 
                 PNMT 
                 0.452623408 
               
               
                   
               
               
                 132 
                 ANMS 
                 0.446947415 
               
               
                   
               
               
                 133 
                 PNVT 
                 0.441515986 
               
               
                   
               
               
                 134 
                 PNHS 
                 0.43719687 
               
               
                   
               
               
                 135 
                 MNVS 
                 0.434864046 
               
               
                   
               
               
                 136 
                 NNIS 
                 0.408557928 
               
               
                   
               
               
                 137 
                 INIT 
                 0.40016673 
               
               
                   
               
               
                 138 
                 VNIT 
                 0.398343399 
               
               
                   
               
               
                 139 
                 FNGS 
                 0.383438044 
               
               
                   
               
               
                 140 
                 VNYS 
                 0.381618237 
               
               
                   
               
               
                 141 
                 TNVS 
                 0.380089456 
               
               
                   
               
               
                 142 
                 GNIT 
                 0.375939233 
               
               
                   
               
               
                 143 
                 VNMT 
                 0.371775726 
               
               
                   
               
               
                 144 
                 INHS 
                 0.359828438 
               
               
                   
               
               
                 145 
                 LNIT 
                 0.358718641 
               
               
                   
               
               
                 146 
                 ANVT 
                 0.351004095 
               
               
                   
               
               
                 147 
                 ANFS 
                 0.346182732 
               
               
                   
               
               
                 148 
                 VNLS 
                 0.341726397 
               
               
                   
               
               
                 149 
                 LNFS 
                 0.339966784 
               
               
                   
               
               
                 150 
                 PNWS 
                 0.334806728 
               
               
                   
               
               
                 151 
                 ANGS 
                 0.332428339 
               
               
                   
               
               
                 152 
                 VNFS 
                 0.325367326 
               
               
                   
               
               
                 153 
                 ANYS 
                 0.321482329 
               
               
                   
               
               
                 154 
                 PNLT 
                 0.317538849 
               
               
                   
               
               
                 155 
                 LNVT 
                 0.307874944 
               
               
                   
               
               
                 156 
                 VNVT 
                 0.300283512 
               
               
                   
               
               
                 157 
                 YNGS 
                 0.291752903 
               
               
                   
               
               
                 158 
                 ANLS 
                 0.279786094 
               
               
                   
               
               
                 159 
                 INIS 
                 0.270744696 
               
               
                   
               
               
                 160 
                 GNVT 
                 0.269651995 
               
               
                   
               
               
                 161 
                 FNHS 
                 0.266061219 
               
               
                   
               
               
                 162 
                 PNRS 
                 0.265194227 
               
               
                   
               
               
                 163 
                 TNIT 
                 0.25866238 
               
               
                   
               
               
                 164 
                 SNMS 
                 0.253083662 
               
               
                   
               
               
                 165 
                 GNLS 
                 0.251946642 
               
               
                   
               
               
                 166 
                 NNIT 
                 0.250115306 
               
               
                   
               
               
                 167 
                 SNIT 
                 0.234106222 
               
               
                   
               
               
                 168 
                 TNMS 
                 0.221310026 
               
               
                   
               
               
                 169 
                 ANHS 
                 0.217808703 
               
               
                   
               
               
                 170 
                 PNRT 
                 0.216307802 
               
               
                   
               
               
                 171 
                 MNIT 
                 0.215031456 
               
               
                   
               
               
                 172 
                 SNVT 
                 0.213276944 
               
               
                   
               
               
                 173 
                 LNMS 
                 0.21284054 
               
               
                   
               
               
                 174 
                 PNHT 
                 0.211595482 
               
               
                   
               
               
                 175 
                 INVT 
                 0.208814402 
               
               
                   
               
               
                 176 
                 VNHS 
                 0.207103204 
               
               
                   
               
               
                 177 
                 NNVS 
                 0.205011148 
               
               
                   
               
               
                 178 
                 LNYS 
                 0.203604231 
               
               
                   
               
               
                 179 
                 VNLT 
                 0.199226218 
               
               
                   
               
               
                 180 
                 LNHT 
                 0.199037207 
               
               
                   
               
               
                 181 
                 ANMT 
                 0.195434618 
               
               
                   
               
               
                 182 
                 GNHS 
                 0.194483535 
               
               
                   
               
               
                 183 
                 FNGT 
                 0.193875356 
               
               
                   
               
               
                 184 
                 PNGT 
                 0.1929585 
               
               
                   
               
               
                 185 
                 SNYS 
                 0.189229065 
               
               
                   
               
               
                 186 
                 LNAT 
                 0.187349711 
               
               
                   
               
               
                 187 
                 PNAT 
                 0.183334397 
               
               
                   
               
               
                 188 
                 PNAS 
                 0.179737178 
               
               
                   
               
               
                 189 
                 SNGS 
                 0.17860807 
               
               
                   
               
               
                 190 
                 YNGT 
                 0.176243637 
               
               
                   
               
               
                 191 
                 MNHT 
                 0.165755541 
               
               
                   
               
               
                 192 
                 GNMS 
                 0.165211568 
               
               
                   
               
               
                 193 
                 VNAT 
                 0.164566376 
               
               
                   
               
               
                 194 
                 VNFT 
                 0.164431729 
               
               
                   
               
               
                 195 
                 FNHT 
                 0.159144461 
               
               
                   
               
               
                 196 
                 ANRS 
                 0.158149197 
               
               
                   
               
               
                 197 
                 LNYT 
                 0.15697147 
               
               
                   
               
               
                 198 
                 TNVT 
                 0.156446198 
               
               
                   
               
               
                 199 
                 GNYS 
                 0.155766768 
               
               
                   
               
               
                 200 
                 GNFS 
                 0.152854107 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 BIN 
                 FREQUENCY 
               
               
                   
               
               
                   
                 0 
                 29 
               
               
                   
               
               
                   
                 0.05 
                 518 
               
               
                   
               
               
                   
                 0.1 
                 45 
               
               
                   
               
               
                   
                 0.15 
                 27 
               
               
                   
               
               
                   
                 0.2 
                 25 
               
               
                   
               
               
                   
                 0.25 
                 12 
               
               
                   
               
               
                   
                 0.3 
                 10 
               
               
                   
               
               
                   
                 0.35 
                 10 
               
               
                   
               
               
                   
                 0.4 
                 9 
               
               
                   
               
               
                   
                 0.45 
                 6 
               
               
                   
               
               
                   
                 0.5 
                 5 
               
               
                   
               
               
                   
                 0.55 
                 12 
               
               
                   
               
               
                   
                 0.6 
                 6 
               
               
                   
               
               
                   
                 0.65 
                 2 
               
               
                   
               
               
                   
                 0.7 
                 2 
               
               
                   
               
               
                   
                 0.75 
                 2 
               
               
                   
               
               
                   
                 0.8 
                 1 
               
               
                   
               
               
                   
                 0.85 
                 1 
               
               
                   
               
               
                   
                 0.9 
                 0 
               
               
                   
               
               
                   
                 0.95 
                 0 
               
               
                   
               
               
                   
                 1 
                 0 
               
               
                   
               
               
                   
                 More 
                 0 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Modification Efficiencies for 4-mer Peptides 
               
               
                 by the N-glycosyltransferase from 
               
               
                 
                   Haemophilus dureyi  
                 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID 
                   
                 MODIFICATION 
               
               
                 NO 
                 SEQUENCE 
                 EFFICIENCY 
               
               
                   
               
               
                 201 
                 ANVT 
                 0.900743 
               
               
                   
               
               
                 202 
                 PNVT 
                 0.888506 
               
               
                   
               
               
                 203 
                 ANIT 
                 0.82341 
               
               
                   
               
               
                 204 
                 PNIT 
                 0.819559 
               
               
                   
               
               
                 205 
                 SNVT 
                 0.7806 
               
               
                   
               
               
                 206 
                 PNAT 
                 0.750574 
               
               
                   
               
               
                 207 
                 ANAT 
                 0.735488 
               
               
                   
               
               
                 208 
                 PNTT 
                 0.662328 
               
               
                   
               
               
                 209 
                 PNMT 
                 0.658102 
               
               
                   
               
               
                 210 
                 ANHT 
                 0.623851 
               
               
                   
               
               
                 211 
                 GNWT 
                 0.623028 
               
               
                   
               
               
                 212 
                 PNST 
                 0.622506 
               
               
                   
               
               
                 213 
                 NNVT 
                 0.612493 
               
               
                   
               
               
                 214 
                 ANST 
                 0.606666 
               
               
                   
               
               
                 215 
                 PNYT 
                 0.605621 
               
               
                   
               
               
                 216 
                 NNAT 
                 0.601921 
               
               
                   
               
               
                 217 
                 ANTT 
                 0.580212 
               
               
                   
               
               
                 218 
                 SNHT 
                 0.573231 
               
               
                   
               
               
                 219 
                 ANYT 
                 0.56827 
               
               
                   
               
               
                 220 
                 ANVS 
                 0.566734 
               
               
                   
               
               
                 221 
                 ANMT 
                 0.558359 
               
               
                   
               
               
                 222 
                 TNVT 
                 0.537524 
               
               
                   
               
               
                 223 
                 PNLT 
                 0.533925 
               
               
                   
               
               
                 224 
                 PNHT 
                 0.524854 
               
               
                   
               
               
                 225 
                 PNFT 
                 0.514359 
               
               
                   
               
               
                 226 
                 TNHT 
                 0.491842 
               
               
                   
               
               
                 227 
                 SNAT 
                 0.485772 
               
               
                   
               
               
                 228 
                 PNRT 
                 0.472573 
               
               
                   
               
               
                 229 
                 ANFT 
                 0.466985 
               
               
                   
               
               
                 230 
                 PNVS 
                 0.45746 
               
               
                   
               
               
                 231 
                 PNIS 
                 0.426112 
               
               
                   
               
               
                 232 
                 ANLT 
                 0.425127 
               
               
                   
               
               
                 233 
                 NNHT 
                 0.413605 
               
               
                   
               
               
                 234 
                 SNIT 
                 0.409056 
               
               
                   
               
               
                 235 
                 ANRT 
                 0.407949 
               
               
                   
               
               
                 236 
                 WNHT 
                 0.398551 
               
               
                   
               
               
                 237 
                 HNVT 
                 0.380896 
               
               
                   
               
               
                 238 
                 GNVT 
                 0.380446 
               
               
                   
               
               
                 239 
                 WNAT 
                 0.366419 
               
               
                   
               
               
                 240 
                 HNHT 
                 0.348211 
               
               
                   
               
               
                 241 
                 SNVS 
                 0.334165 
               
               
                   
               
               
                 242 
                 WNVT 
                 0.323861 
               
               
                   
               
               
                 243 
                 VNVT 
                 0.315139 
               
               
                   
               
               
                 244 
                 VNAT 
                 0.304739 
               
               
                   
               
               
                 245 
                 TNAT 
                 0.304363 
               
               
                   
               
               
                 246 
                 TNST 
                 0.2587 
               
               
                   
               
               
                 247 
                 ANHS 
                 0.254992 
               
               
                   
               
               
                 248 
                 VNIT 
                 0.242711 
               
               
                   
               
               
                 249 
                 ANIS 
                 0.241908 
               
               
                   
               
               
                 250 
                 GNHT 
                 0.232812 
               
               
                   
               
               
                 251 
                 NNRT 
                 0.231829 
               
               
                   
               
               
                 252 
                 NNTT 
                 0.229518 
               
               
                   
               
               
                 253 
                 SNTT 
                 0.22013 
               
               
                   
               
               
                 254 
                 MNVT 
                 0.214801 
               
               
                   
               
               
                 255 
                 SNST 
                 0.214257 
               
               
                   
               
               
                 256 
                 VNMT 
                 0.204642 
               
               
                   
               
               
                 257 
                 TNIT 
                 0.19798 
               
               
                   
               
               
                 258 
                 NNST 
                 0.187755 
               
               
                   
               
               
                 259 
                 MNIT 
                 0.17966 
               
               
                   
               
               
                 260 
                 WNRT 
                 0.179016 
               
               
                   
               
               
                 261 
                 SNRT 
                 0.178091 
               
               
                   
               
               
                 262 
                 GNIT 
                 0.17171 
               
               
                   
               
               
                 263 
                 MNHT 
                 0.168865 
               
               
                   
               
               
                 264 
                 HNAT 
                 0.162251 
               
               
                   
               
               
                 265 
                 SNMT 
                 0.157808 
               
               
                   
               
               
                 266 
                 PNHS 
                 0.156218 
               
               
                   
               
               
                 267 
                 SNYT 
                 0.152193 
               
               
                   
               
               
                 268 
                 ANGT 
                 0.138123 
               
               
                   
               
               
                 269 
                 ENST 
                 0.137658 
               
               
                   
               
               
                 270 
                 TNNT 
                 0.136027 
               
               
                   
               
               
                 271 
                 PNMS 
                 0.132182 
               
               
                   
               
               
                 272 
                 TNTT 
                 0.13073 
               
               
                   
               
               
                 273 
                 PNGT 
                 0.130558 
               
               
                   
               
               
                 274 
                 VNST 
                 0.123327 
               
               
                   
               
               
                 275 
                 LNVT 
                 0.119945 
               
               
                   
               
               
                 276 
                 GNAT 
                 0.119761 
               
               
                   
               
               
                 277 
                 PNYS 
                 0.118633 
               
               
                   
               
               
                 278 
                 PNKT 
                 0.118554 
               
               
                   
               
               
                 279 
                 ENVT 
                 0.113663 
               
               
                   
               
               
                 280 
                 VNHT 
                 0.109097 
               
               
                   
               
               
                 281 
                 NNHS 
                 0.107909 
               
               
                   
               
               
                 282 
                 ENAT 
                 0.105764 
               
               
                   
               
               
                 283 
                 SNGT 
                 0.105569 
               
               
                   
               
               
                 284 
                 SNHS 
                 0.104335 
               
               
                   
               
               
                 285 
                 NNYT 
                 0.103967 
               
               
                   
               
               
                 286 
                 MNAT 
                 0.103538 
               
               
                   
               
               
                 287 
                 PNTS 
                 0.09756 
               
               
                   
               
               
                 288 
                 ANAS 
                 0.095155 
               
               
                   
               
               
                 289 
                 GNYT 
                 0.093017 
               
               
                   
               
               
                 290 
                 LNAT 
                 0.092369 
               
               
                   
               
               
                 291 
                 NNVS 
                 0.088442 
               
               
                   
               
               
                 292 
                 ANQT 
                 0.088372 
               
               
                   
               
               
                 293 
                 TNVS 
                 0.087944 
               
               
                   
               
               
                 294 
                 PNAS 
                 0.086776 
               
               
                   
               
               
                 295 
                 GNVS 
                 0.086098 
               
               
                   
               
               
                 296 
                 PNQT 
                 0.086057 
               
               
                   
               
               
                 297 
                 ANKT 
                 0.0857 
               
               
                   
               
               
                 298 
                 SNLT 
                 0.085519 
               
               
                   
               
               
                 299 
                 TNMT 
                 0.084772 
               
               
                   
               
               
                 300 
                 TNHS 
                 0.082042 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 BIN 
                 FREQUENCY 
               
               
                   
               
               
                   
                 0 
                 61 
               
               
                   
               
               
                   
                 0.05 
                 529 
               
               
                   
               
               
                   
                 0.1 
                 46 
               
               
                   
               
               
                   
                 0.15 
                 19 
               
               
                   
               
               
                   
                 0.2 
                 11 
               
               
                   
               
               
                   
                 0.25 
                 9 
               
               
                   
               
               
                   
                 0.3 
                 2 
               
               
                   
               
               
                   
                 0.35 
                 6 
               
               
                   
               
               
                   
                 0.4 
                 4 
               
               
                   
               
               
                   
                 0.45 
                 5 
               
               
                   
               
               
                   
                 0.5 
                 5 
               
               
                   
               
               
                   
                 0.55 
                 4 
               
               
                   
               
               
                   
                 0.6 
                 5 
               
               
                   
               
               
                   
                 0.65 
                 7 
               
               
                   
               
               
                   
                 0.7 
                 2 
               
               
                   
               
               
                   
                 0.75 
                 1 
               
               
                   
               
               
                   
                 0.8 
                 2 
               
               
                   
               
               
                   
                 0.85 
                 2 
               
               
                   
               
               
                   
                 0.9 
                 1 
               
               
                   
               
               
                   
                 0.95 
                 1 
               
               
                   
               
               
                   
                 1 
                 0 
               
               
                   
               
               
                   
                 More 
                 0 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Modification Efficiencies for 4-mer Peptides 
               
               
                 by the N-glycosyltransferase from 
               
               
                 
                   Mannheimia haemolytica  
                 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID 
                   
                 MODIFICATION 
               
               
                 NO 
                 SEQUENCE 
                 EFFICIENCY 
               
               
                   
               
               
                 301 
                 ANYT 
                 0.96465 
               
               
                   
               
               
                 302 
                 ANVT 
                 0.958746 
               
               
                   
               
               
                 303 
                 SNVT 
                 0.930745 
               
               
                   
               
               
                 304 
                 PNVT 
                 0.920004 
               
               
                   
               
               
                 305 
                 WNVT 
                 0.908308 
               
               
                   
               
               
                 306 
                 GNWT 
                 0.882545 
               
               
                   
               
               
                 307 
                 PNMT 
                 0.87976 
               
               
                   
               
               
                 308 
                 ANIT 
                 0.878841 
               
               
                   
               
               
                 309 
                 WNAT 
                 0.878522 
               
               
                   
               
               
                 310 
                 NNVT 
                 0.873659 
               
               
                   
               
               
                 311 
                 ANAT 
                 0.873087 
               
               
                   
               
               
                 312 
                 PNIT 
                 0.868744 
               
               
                   
               
               
                 313 
                 ANFT 
                 0.866228 
               
               
                   
               
               
                 314 
                 PNAT 
                 0.86522 
               
               
                   
               
               
                 315 
                 PNYT 
                 0.8592 
               
               
                   
               
               
                 316 
                 PNFT 
                 0.858603 
               
               
                   
               
               
                 317 
                 ANRT 
                 0.853493 
               
               
                   
               
               
                 318 
                 SNAT 
                 0.850271 
               
               
                   
               
               
                 319 
                 HNVT 
                 0.844279 
               
               
                   
               
               
                 320 
                 GNVT 
                 0.843273 
               
               
                   
               
               
                 321 
                 PNST 
                 0.839726 
               
               
                   
               
               
                 322 
                 PNRT 
                 0.838078 
               
               
                   
               
               
                 323 
                 TNVT 
                 0.835131 
               
               
                   
               
               
                 324 
                 SNHT 
                 0.83346 
               
               
                   
               
               
                 325 
                 PNTT 
                 0.830543 
               
               
                   
               
               
                 326 
                 ANST 
                 0.824501 
               
               
                   
               
               
                 327 
                 PNLT 
                 0.823914 
               
               
                   
               
               
                 328 
                 ANMT 
                 0.821819 
               
               
                   
               
               
                 329 
                 NNAT 
                 0.817938 
               
               
                   
               
               
                 330 
                 ANTT 
                 0.811151 
               
               
                   
               
               
                 331 
                 WNHT 
                 0.810613 
               
               
                   
               
               
                 332 
                 NNRT 
                 0.810094 
               
               
                   
               
               
                 333 
                 WNRT 
                 0.80212 
               
               
                   
               
               
                 334 
                 ANHT 
                 0.787241 
               
               
                   
               
               
                 335 
                 MNIT 
                 0.764916 
               
               
                   
               
               
                 336 
                 MNHT 
                 0.756672 
               
               
                   
               
               
                 337 
                 SNVS 
                 0.753741 
               
               
                   
               
               
                 338 
                 SNIT 
                 0.753394 
               
               
                   
               
               
                 339 
                 HNHT 
                 0.74824 
               
               
                   
               
               
                 340 
                 GNHT 
                 0.743891 
               
               
                   
               
               
                 341 
                 TNHT 
                 0.739773 
               
               
                   
               
               
                 342 
                 WNIT 
                 0.738131 
               
               
                   
               
               
                 343 
                 TNAT 
                 0.733502 
               
               
                   
               
               
                 344 
                 MNVT 
                 0.73061 
               
               
                   
               
               
                 345 
                 ANLT 
                 0.724314 
               
               
                   
               
               
                 346 
                 SNRT 
                 0.722217 
               
               
                   
               
               
                 347 
                 NNHT 
                 0.719883 
               
               
                   
               
               
                 348 
                 VNVT 
                 0.710196 
               
               
                   
               
               
                 349 
                 MNAT 
                 0.706852 
               
               
                   
               
               
                 350 
                 PNHT 
                 0.69745 
               
               
                   
               
               
                 351 
                 HNAT 
                 0.688339 
               
               
                   
               
               
                 352 
                 SNYT 
                 0.687115 
               
               
                   
               
               
                 353 
                 VNAT 
                 0.672586 
               
               
                   
               
               
                 354 
                 ANVS 
                 0.672556 
               
               
                   
               
               
                 355 
                 ENVT 
                 0.663018 
               
               
                   
               
               
                 356 
                 SNTT 
                 0.661821 
               
               
                   
               
               
                 357 
                 NNST 
                 0.660101 
               
               
                   
               
               
                 358 
                 SNST 
                 0.648362 
               
               
                   
               
               
                 359 
                 VNMT 
                 0.644028 
               
               
                   
               
               
                 360 
                 NNTT 
                 0.641425 
               
               
                   
               
               
                 361 
                 TNST 
                 0.624424 
               
               
                   
               
               
                 362 
                 PNVS 
                 0.624348 
               
               
                   
               
               
                 363 
                 HNRT 
                 0.612555 
               
               
                   
               
               
                 364 
                 WNST 
                 0.610847 
               
               
                   
               
               
                 365 
                 ENST 
                 0.59463 
               
               
                   
               
               
                 366 
                 ENRT 
                 0.584351 
               
               
                   
               
               
                 367 
                 SNMT 
                 0.581497 
               
               
                   
               
               
                 368 
                 HNIT 
                 0.57906 
               
               
                   
               
               
                 369 
                 GNYT 
                 0.574794 
               
               
                   
               
               
                 370 
                 WNMT 
                 0.560978 
               
               
                   
               
               
                 371 
                 WNTT 
                 0.558826 
               
               
                   
               
               
                 372 
                 INVT 
                 0.554687 
               
               
                   
               
               
                 373 
                 ENAT 
                 0.554528 
               
               
                   
               
               
                 374 
                 ANQT 
                 0.550673 
               
               
                   
               
               
                 375 
                 GNAT 
                 0.549155 
               
               
                   
               
               
                 376 
                 MNLT 
                 0.549058 
               
               
                   
               
               
                 377 
                 TNVS 
                 0.546237 
               
               
                   
               
               
                 378 
                 PNQT 
                 0.545224 
               
               
                   
               
               
                 379 
                 MNRT 
                 0.545093 
               
               
                   
               
               
                 380 
                 NNYT 
                 0.540375 
               
               
                   
               
               
                 381 
                 SNFT 
                 0.53759 
               
               
                   
               
               
                 382 
                 LNVT 
                 0.53582 
               
               
                   
               
               
                 383 
                 PNIS 
                 0.530673 
               
               
                   
               
               
                 384 
                 QNVT 
                 0.523167 
               
               
                   
               
               
                 385 
                 VNST 
                 0.522874 
               
               
                   
               
               
                 386 
                 VNIT 
                 0.521194 
               
               
                   
               
               
                 387 
                 GNIT 
                 0.511218 
               
               
                   
               
               
                 388 
                 ANIS 
                 0.507664 
               
               
                   
               
               
                 389 
                 HNTT 
                 0.495843 
               
               
                   
               
               
                 390 
                 YNVT 
                 0.490232 
               
               
                   
               
               
                 391 
                 FNVT 
                 0.486868 
               
               
                   
               
               
                 392 
                 VNRT 
                 0.47939 
               
               
                   
               
               
                 393 
                 WNYT 
                 0.476276 
               
               
                   
               
               
                 394 
                 ANWT 
                 0.474853 
               
               
                   
               
               
                 395 
                 LNAT 
                 0.472108 
               
               
                   
               
               
                 396 
                 ENHT 
                 0.469099 
               
               
                   
               
               
                 397 
                 ANGT 
                 0.463129 
               
               
                   
               
               
                 398 
                 MNST 
                 0.461919 
               
               
                   
               
               
                 399 
                 TNIT 
                 0.461009 
               
               
                   
               
               
                 400 
                 LNRT 
                 0.457687 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 BIN 
                 FREQUENCY 
               
               
                   
               
               
                   
                 0 
                 11 
               
               
                   
               
               
                   
                 0.05 
                 431 
               
               
                   
               
               
                   
                 0.1 
                 49 
               
               
                   
               
               
                   
                 0.15 
                 22 
               
               
                   
               
               
                   
                 0.2 
                 27 
               
               
                   
               
               
                   
                 0.25 
                 21 
               
               
                   
               
               
                   
                 0.3 
                 15 
               
               
                   
               
               
                   
                 0.35 
                 13 
               
               
                   
               
               
                   
                 0.4 
                 15 
               
               
                   
               
               
                   
                 0.45 
                 17 
               
               
                   
               
               
                   
                 0.5 
                 13 
               
               
                   
               
               
                   
                 0.55 
                 14 
               
               
                   
               
               
                   
                 0.6 
                 10 
               
               
                   
               
               
                   
                 0.65 
                 7 
               
               
                   
               
               
                   
                 0.7 
                 8 
               
               
                   
               
               
                   
                 0.75 
                 11 
               
               
                   
               
               
                   
                 0.8 
                 5 
               
               
                   
               
               
                   
                 0.85 
                 15 
               
               
                   
               
               
                   
                 0.9 
                 13 
               
               
                   
               
               
                   
                 0.95 
                 3 
               
               
                   
               
               
                   
                 1 
                 2 
               
               
                   
               
               
                   
                 More 
                 0 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Modification Efficiencies for 4-mer Peptides 
               
               
                 by the N-glycosyltransferase from 
               
               
                 
                   Haemophilus influenza  
                 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID 
                   
                 MODIFICATION 
               
               
                 NO 
                 SEQUENCE 
                 EFFICIENCY 
               
               
                   
               
               
                 401 
                 PNIT 
                 0.872492503 
               
               
                   
               
               
                 402 
                 ANAT 
                 0.857052653 
               
               
                   
               
               
                 403 
                 PNVT 
                 0.795394559 
               
               
                   
               
               
                 404 
                 PNMT 
                 0.718961169 
               
               
                   
               
               
                 405 
                 PNAT 
                 0.603312989 
               
               
                   
               
               
                 406 
                 PNLT 
                 0.590978057 
               
               
                   
               
               
                 407 
                 ANVT 
                 0.58963897 
               
               
                   
               
               
                 408 
                 ANMT 
                 0.588036414 
               
               
                   
               
               
                 409 
                 ANIT 
                 0.570257163 
               
               
                   
               
               
                 410 
                 HNVT 
                 0.554514307 
               
               
                   
               
               
                 411 
                 PNFT 
                 0.464502861 
               
               
                   
               
               
                 412 
                 ANFT 
                 0.461944104 
               
               
                   
               
               
                 413 
                 PNVS 
                 0.442701706 
               
               
                   
               
               
                 414 
                 PNYT 
                 0.433228465 
               
               
                   
               
               
                 415 
                 MNIT 
                 0.432644327 
               
               
                   
               
               
                 416 
                 PNRT 
                 0.421151455 
               
               
                   
               
               
                 417 
                 ANRT 
                 0.41573846 
               
               
                   
               
               
                 418 
                 PNIS 
                 0.392207849 
               
               
                   
               
               
                 419 
                 SNVT 
                 0.38329666 
               
               
                   
               
               
                 420 
                 NNAT 
                 0.381109213 
               
               
                   
               
               
                 421 
                 ANVS 
                 0.3782757 
               
               
                   
               
               
                 422 
                 VNMT 
                 0.373309945 
               
               
                   
               
               
                 423 
                 INVT 
                 0.373104823 
               
               
                   
               
               
                 424 
                 ANHT 
                 0.355689212 
               
               
                   
               
               
                 425 
                 NNVT 
                 0.336341478 
               
               
                   
               
               
                 426 
                 PNHT 
                 0.329569969 
               
               
                   
               
               
                 427 
                 HNHT 
                 0.321026995 
               
               
                   
               
               
                 428 
                 WNVT 
                 0.311422801 
               
               
                   
               
               
                 429 
                 ANLT 
                 0.295331218 
               
               
                   
               
               
                 430 
                 PNTT 
                 0.279773705 
               
               
                   
               
               
                 431 
                 MNVT 
                 0.257204301 
               
               
                   
               
               
                 432 
                 ANYT 
                 0.251127086 
               
               
                   
               
               
                 433 
                 MNLT 
                 0.247786488 
               
               
                   
               
               
                 434 
                 VNIT 
                 0.24240979 
               
               
                   
               
               
                 435 
                 PNST 
                 0.226010829 
               
               
                   
               
               
                 436 
                 WNAT 
                 0.221585505 
               
               
                   
               
               
                 437 
                 FNAT 
                 0.221034299 
               
               
                   
               
               
                 438 
                 ANIS 
                 0.20792148 
               
               
                   
               
               
                 439 
                 TNVT 
                 0.190595546 
               
               
                   
               
               
                 440 
                 INMT 
                 0.179126526 
               
               
                   
               
               
                 441 
                 HNVS 
                 0.177090897 
               
               
                   
               
               
                 442 
                 HNRT 
                 0.167363107 
               
               
                   
               
               
                 443 
                 PNLS 
                 0.165777071 
               
               
                   
               
               
                 444 
                 PNMS 
                 0.1617613 
               
               
                   
               
               
                 445 
                 VNVT 
                 0.160963364 
               
               
                   
               
               
                 446 
                 ANST 
                 0.159600972 
               
               
                   
               
               
                 447 
                 HNAT 
                 0.157573446 
               
               
                   
               
               
                 448 
                 ANTT 
                 0.141177856 
               
               
                   
               
               
                 449 
                 WNHT 
                 0.14031522 
               
               
                   
               
               
                 450 
                 NNHT 
                 0.140082578 
               
               
                   
               
               
                 451 
                 PNYS 
                 0.136122223 
               
               
                   
               
               
                 452 
                 HNIT 
                 0.134601969 
               
               
                   
               
               
                 453 
                 SNHT 
                 0.134282522 
               
               
                   
               
               
                 454 
                 TNHT 
                 0.127849126 
               
               
                   
               
               
                 455 
                 GNWT 
                 0.126267091 
               
               
                   
               
               
                 456 
                 SNVS 
                 0.126264092 
               
               
                   
               
               
                 457 
                 FNVT 
                 0.121700545 
               
               
                   
               
               
                 458 
                 SNAT 
                 0.121390895 
               
               
                   
               
               
                 459 
                 VNAT 
                 0.117290489 
               
               
                   
               
               
                 460 
                 PNKT 
                 0.110401803 
               
               
                   
               
               
                 461 
                 NNRT 
                 0.106761729 
               
               
                   
               
               
                 462 
                 WNMT 
                 0.102229077 
               
               
                   
               
               
                 463 
                 VNRT 
                 0.095070318 
               
               
                   
               
               
                 464 
                 LNLT 
                 0.095068093 
               
               
                   
               
               
                 465 
                 PNHS 
                 0.094654507 
               
               
                   
               
               
                 466 
                 LNMT 
                 0.089964838 
               
               
                   
               
               
                 467 
                 HNMT 
                 0.089441554 
               
               
                   
               
               
                 468 
                 ANHS 
                 0.089162334 
               
               
                   
               
               
                 469 
                 INIT 
                 0.082852649 
               
               
                   
               
               
                 470 
                 HNTT 
                 0.080023423 
               
               
                   
               
               
                 471 
                 SNIT 
                 0.079360519 
               
               
                   
               
               
                 472 
                 YNRT 
                 0.079177876 
               
               
                   
               
               
                 473 
                 INTVS 
                 0.077276724 
               
               
                   
               
               
                 474 
                 YNVT 
                 0.076140663 
               
               
                   
               
               
                 475 
                 SNYT 
                 0.074808805 
               
               
                   
               
               
                 476 
                 LNRT 
                 0.071432067 
               
               
                   
               
               
                 477 
                 NNMT 
                 0.071361253 
               
               
                   
               
               
                 478 
                 LNVT 
                 0.069067056 
               
               
                   
               
               
                 479 
                 HNHS 
                 0.068650466 
               
               
                   
               
               
                 480 
                 ANKT 
                 0.066420398 
               
               
                   
               
               
                 481 
                 ANYS 
                 0.065983037 
               
               
                   
               
               
                 482 
                 VNLT 
                 0.065024642 
               
               
                   
               
               
                 483 
                 TNAT 
                 0.064442386 
               
               
                   
               
               
                 484 
                 LNIT 
                 0.062291989 
               
               
                   
               
               
                 485 
                 PNQT 
                 0.06199128 
               
               
                   
               
               
                 486 
                 LNAT 
                 0.060627653 
               
               
                   
               
               
                 487 
                 ANLS 
                 0.060442633 
               
               
                   
               
               
                 488 
                 FNHT 
                 0.060271 
               
               
                   
               
               
                 489 
                 NNVS 
                 0.060003436 
               
               
                   
               
               
                 490 
                 PNFS 
                 0.059684221 
               
               
                   
               
               
                 491 
                 GNVT 
                 0.058773849 
               
               
                   
               
               
                 492 
                 ANQT 
                 0.057167365 
               
               
                   
               
               
                 493 
                 RNHT 
                 0.054084953 
               
               
                   
               
               
                 494 
                 PNTS 
                 0.052231141 
               
               
                   
               
               
                 495 
                 WNVS 
                 0.051913903 
               
               
                   
               
               
                 496 
                 ANAS 
                 0.051662869 
               
               
                   
               
               
                 497 
                 MNAT 
                 0.050851556 
               
               
                   
               
               
                 498 
                 HNYT 
                 0.04898975 
               
               
                   
               
               
                 499 
                 SNLT 
                 0.048372175 
               
               
                   
               
               
                 500 
                 TNIT 
                 0.048052479 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 BIN 
                 FREQUENCY 
               
               
                   
               
               
                   
                 0 
                 99 
               
               
                   
               
               
                   
                 0.05 
                 526 
               
               
                   
               
               
                   
                 0.1 
                 35 
               
               
                   
               
               
                   
                 0.15 
                 15 
               
               
                   
               
               
                   
                 0.2 
                 9 
               
               
                   
               
               
                   
                 0.25 
                 6 
               
               
                   
               
               
                   
                 0.3 
                 4 
               
               
                   
               
               
                   
                 0.35 
                 4 
               
               
                   
               
               
                   
                 0.4 
                 7 
               
               
                   
               
               
                   
                 0.45 
                 5 
               
               
                   
               
               
                   
                 0.5 
                 2 
               
               
                   
               
               
                   
                 0.55 
                 0 
               
               
                   
               
               
                   
                 0.6 
                 5 
               
               
                   
               
               
                   
                 0.65 
                 1 
               
               
                   
               
               
                   
                 0.7 
                 0 
               
               
                   
               
               
                   
                 0.75 
                 1 
               
               
                   
               
               
                   
                 0.8 
                 1 
               
               
                   
               
               
                   
                 0.85 
                 0 
               
               
                   
               
               
                   
                 0.9 
                 2 
               
               
                   
               
               
                   
                 0.95 
                 0 
               
               
                   
               
               
                   
                 1 
                 0 
               
               
                   
               
               
                   
                 More 
                 0 
               
               
                   
               
            
           
         
       
     
     Example 4—Site-Specific Control of Multiple Glycosylation Sites Using Unique Enzyme Specificities 
     Introduction 
     Glycosylation, the attachment of sugar moieties to amino acid side-chains, is one of the most common post-translational modifications found in nature 1  and is known to endow proteins with new functions and profoundly affect stability, potency, and half-life of protein therapeutics 3, 4, 6 . However, glycoproteins derived from living cells are usually a complex mixture of glycosylation structures at varying levels at multiple glycosylation sites 3 . This complexity is one of the core challenges to the systematic understanding of the activity and properties of specific glycoforms (bearing specific glycosylation structures at specific points within proteins) and therefore the development and optimization of glycoproteins for biotechnological applications 3 . While significant advances have been made in glycoengineering bacterial 8 , yeast 12 , and mammalian 11  cells for more homogeneous glycoprotein expression, a generalizable technique for obtaining user-defined glycoforms from cells remains elusive 60 . 
     New developments in chemical and chemoenzymatic methods for in vitro construction of homogeneous glycoproteins have enabled the synthesis and study of diverse glycoproteins with rigorously defined glycan structures 60 . For example, total chemical synthesis has been used to produce human EPO and test the function of each glycan by assembling constituent peptides and glycopeptides 61, 62 . However, total chemical synthesis is very costly, requires specialized expertise, and is difficult for large proteins 60 . Recently, great strides have been made in the use of chemo-enzymatic methods to remodel or install homogeneous glycans at monosaccharide modified proteins 60 . The Wang group has developed a suite of endoglycosidases to remodel glycans and used them to carefully study the function of human antibodies with defined glycosylation structures 60 , these enzymes have been used to find that the S2G2 or G2 modification provides the most efficient antibody dependent cell-killing 6, 63  The Davis group and others have combined this method with the incorporation of non-standard amino acids and modification of cysteine residues to install glycans using site-directed mutagenesis 64-67 . 
     However, because only one unique non-standard amino acid can be reliably incorporated into a protein and current chemoenzymatic methods cannot distinguish between modification sites which are chemically nearly identical 60 , they are limited to the synthesis of proteins with the same, at most two, glycosylation structures at all sites 60, 68 . In contrast, glycoproteins often contain multiple glycosylation sites with distinct glycosylation structures at each position which can interact synergistically to effect protein function 65, 69-71. New methods are needed to site-specifically control glycosylation so that glycoproteins with defined combinations of glycans and the interactions between them can be studied and optimized to engineer precise or multifunctional glycoprotein therapeutics and vaccines 60 . A set of glycosyltransferases which could distinguish between multiple glycosylation sites and make the first monosaccharide modification would permit the controlled construction of each glycosylation site afterwards by well-established chemoenzymatic methods in a sequential fashion. However, due to insufficient characterization of glycosyltransferase peptide substrate preferences, such a system has not been realized. 
     Here we report a strategy to site-specifically control the glycosylation of up to four sites within a single target, which we developed by discovering and rigorously characterizing the unique peptide acceptor specificities of N-linked glycosyltransferase (NGT) enzymes ( FIG. 23 ). Because existing characterization of N-linked glycosyltransferases are insufficient to enable the selection of specifically interacting sequence-enzyme pairs within the canonical N-linked glycosylation sequences N-X-S/T-X (where X is not P), we employed our recently developed method called “GlycoSCORES” to rapidly test the activity of 41 putative N-linked GTs and rigorously characterize the activities of three NGTs, combining with a ApNGT mutants, exhibiting strong activity and differences in peptide specificity. We then optimized 6-mer peptide sequences (called GlycTags) which are differentially modified by these four enzymes. We show that when these GlycTags are placed into a single target protein, glycosylation can be site-specifically controlled at up to four sites by the sequential addition of specific NGTs. When combined with existing technologies for chemo-enzymatic elaboration of single monosaccharide to homogeneous glycans, our method for site-specific control of glycosylation could significantly advance our understanding of how multiple glycans interact within a single protein and our ability to engineer glycoproteins for specific purposes. 
     Results 
     Phylogenetic screening for NGT activity. In order to find NGTs that may possess different peptide preferences and could enable specific targeting of multiple glycosylation sites within a single protein, we sought out previously uncharacterized NGT homologs. We performed a phylogenetic analysis of the CAZY database family 41 14 , which is known to contain N-linked polypeptide glucosyltransferases (NGTs) and O-linked N-Acetylglucosaminyltransferases (OGTs). From this phylogenetic analysis, we selected 41 putative N-linked glycosyltransferases from bacteria. In selecting enzymes for our screen, we sought to balance sequence diversity with likelihood of possessing NGT activity by selecting enzymes that are both closely and distantly related to previously characterized NGTs, such as ApNGT. 
     Site-specific control of glycosylation by rapid enzyme characterization and sequential addition of enzymes. Four NGT homologs were selected from the phylogenetic screen of putative NGT enzymes and characterized using GlycoSCORES to find differences in peptide specificity. (See  FIG. 23 ). Peptide sequences showing conditional orthogonality which would enable site-specific glycosylation when applied in the correct order, were discovered by further GlycoSCORES optimization. Optimized GlycTags were incorporated into a single glycoprotein and NGTs were added sequentially to site-specifically control glycosylation at up to four glycosylation sites within one protein. Sugars, modification sites, and NGTs have been color coded for illustration purposes. 
     GlycoSCORES screening of NGT homologs for unique peptide activities. Six representative N-linked glycosylation peptide substrates were screened with 41 putative GTs from CAZY database (GT family 41). (See  FIG. 24 ). The phylogenetic tree uses human OGT as the outgroup due to its sequence divergence and eukaryotic origin. Six enzymes were found to have NGT activity, with strong activity ( Actinobacillus pleuropneumoniae  NGT (ApNGT),  Escherichia coli  NGT (EcNGT),  Haemophilus  influenza NGT (HiNGT),  Mannheimia haemolytica  NGT (MhNGT), and  Haemophilus dureyi  NGT (HdNGT)). GlycoSCORES screening was performing similarly as disclosed in Examples 1 and 2. NGTs were produced in CFPS and mixed with UDP-glucose sugar donor and cysteine-containing peptide substrates which were immobilized to a maleimide self-assembled monolayer and characterized by SAMDI-MS. Some NGTs were found to have differences in peptide selectivity (MS spectra). In particular, the specificity of HiNGT, EcNGT, ApNGT and ApNGT Q469A  for a peptide substrate library of the form X −1 NX +1 TRC was analyzed. These four NGTs showed differences in peptide selectivity from each other, but not from MhNGT or HdNGT (data not shown). Experimental conditions: 0.42 μM CFPS HiNGT or 0.75 μM CFPS EcNGT, 30° C. for 21 h; 0.055 μM CFPS ApNGT or 0.014 μM CFPS ApNGT Q469A , 30° C. for 1 h. Expression of HiNGT, EcNGT, ApNGT and ApNGT Q469A  by CFPS was verified (data not shown). 
     Optimization of Differentially Modified Sequences to Obtain Conditionally Orthogonal GlycTags 
     A peptide library was generated in order to identify optimally conditional orthogonal GlycTag sequences for HiNGT, EcNGT, ApNGT and ApNGT Q469A . The generated library generally had the sequence X −1 NX +1 (T/S)RC where X −1  and X +1  independently were any amino acid. The C-terminal cysteine of the peptide library was used to immobilize the peptides on a SAMDI plate for GlycoSCORE analysis and screening as described in Examples 1 and 2. The analysis and screening revealed that each of HiNGT, EcNGT, ApNGT and ApNGT Q469A  exhibited differential sequence preferences in regard to N-glycosylation (e.g., FNQT (SEQ ID NO:520), YNLT (SEQ ID NO:521), YNRT (SEQ ID NO:472), INWT (SEQ ID NO:522), WNWT (SEQ ID NO:523), INQT (SEQ ID NO:524) only for ApNGT Q469A ; ENVT (SEQ ID NOs:25,279,355) for ApNGT but not for EcNGT or HiNGT; WNGS (SEQ ID NO:110), LNHS (SEQ ID NO:119), and GNIS (SEQ ID NO:111) for EcNGT but not for HiNGT; and PNLT (SEQ ID NOs:45,154,223,327,406), ANVT (SEQ ID NOs:1,146,201,302,407), PNIT (SEQ ID NOs:10,108,204,312,401) and PNVT (SEQ ID NOs:2, 133,202,304,403) for HiNGT). 
     Selected sequences with differential modification patterns were resynthesized and screened with 19 amino acids in the X −2  position in an X-member X −2 (X −1 NX +1 T/S)RC library using all four enzymes. Several sequences that exhibited differential modification patterns between the four enzymes were identified (e.g., AFNQT (SEQ ID NO:525), SYNLT (SEQ ID NO:526), AYNLT (SEQ ID NO:527), DFNQT (SEQ ID NO:528), SFNQT (SEQ ID NO:529), and DYNLT (SEQ ID NO:530) only for ApNGT Q469A ; NENVT (SEQ ID NO:531) for ApNGT but not for EcNGT or HiNGT; FGNWS (SEQ ID NO:531), WGNWS (SEQ ID NO:532), FGNIS (SEQ ID NO:533), YGNWS (SEQ ID NO:534), TGNIS (SEQ ID NO:535), LGNIS (SEQ ID NO:536), AGNIS (SEQ ID NO:537), VGNIS (SEQ ID NO:538), MGNIS (SEQ ID NO:539), and IGNIS (SEQ ID NO:540) for EcNGT but not for HiNGT; and YGNWT (SEQ ID NO:541), WPNLT (SEQ ID NO:542), WGNWT (SEQ ID NO:543), WPNIT (SEQ ID NO:544), and WPNVT (SEQ ID NO:545) for HiNGT). 
     Selected peptide sequences again were resynthesized and screened with 19 amino acids in the X −3  positions in an X-member X −3 (X −2 (X −1 NX +1 T/S)RC library using all four NGTs. Several sequences that exhibited differential modification patterns between the four enzymes were identified (e.g., WDYNLT (SEQ ID NO:546) only for ApNGT Q469A ; LNENVT (SEQ ID NO:547) for ApNGT but not for EcNGT or HiNGT; YMGNIS (SEQ ID NO:548) for EcNGT but not for HiNGT; and WYANVT (SEQ ID NO:549) for HiNGT). 
     Optimized GlycTag sequences show conditional orthogonality at peptide level and enable differential targeting of glycosylation sites within protein. Conditional orthogonality of optimized 6-mer GlycTags. Selected GlycTags were screened for HiNGT, EcNGT, ApNGT and ApNGT Q469A  modification by SAMDI in triplicate experiments. (See  FIG. 25 a   ). Optimized 6-mer GlycTags were inserted into the N-terminus, C-terminus, and two exposed loops of the glycosylation model protein Im7, with flanking sequences of RATT (SEQ ID NO:516)-GlycTag-AGGR (SEQ ID NO:517) to facilitate trypsinization and quantitative LC-MS analysis. (See  FIG. 25 b   ). Differential targeting of four optimized GlycTags within a single Im7 target protein. Im7 bearing the four optimized GlycTags was reacted with 2.5 mM UDP-Glucose and various concentrations of each purified NGT for 4 hrs. After the modification, Im7 was purified using Ni-NTA functionalized magnetic beads, treated with trypsin and analyzed by LC-qTOF. (See  FIG. 25 c   ). Conditional orthogonality of each NGT for 6-mer GlycTags within Im7 under optimized conditions was observed. (See  FIG. 25 d   ). 
     Site-specific control of glycosylation at four distinct GlycTag sequences within one target protein. Site-specific control of glycosylation at the four distinct GlycTag sequences within one target protein was tested and observed. (See  FIGS. 26 a  and 26 b   ). 
     Discussion 
     Here, we report the discovery of unique N-glucosyltransferases (NGTs) and specificities for corresponding peptide targets (GlycTag) and optimization of conditionally orthogonal NGT-GlycTag pairs. Importantly, we demonstrate that conditional orthogonality behavior observed at the peptide level was also achieved at the protein level. We also demonstrate a system and workflow for site-specific control using four different NGTs and four different GlycTags when the different GlycTags were fused into a protein at four locations. Now that we have overcome the critical challenge of site-specificity, our sequential glycosylation technique can be combined with well-established chemo-enzymatic methods to install multiple, distinct N-linked glycans onto a single protein using endoglycosidases or other glycosyltranferases during each modification step. Future work will be directed to rigorously characterizing ApNGT mutants to expand the available repertoire of enzymes and develop more highly orthogonal NGT and GlycTag pairs. 
     Now that we have overcome the challenge of site-specificity, our sequential glycosylation technique can be combined with well-established chemo-enzymatic methods 60  to install multiple, distinct N-linked glycans onto a single protein using endoglycosidases or other glycosyltranferases during each modification step. With the depth of characterization data found in this work and our continuing efforts to rigorously understand NGT specificities, we can modify any therapeutic target protein with multiple glycans with minimal change of the amino acid sequence by strategically using known enzymes with specific activities. Towards this goal, we are currently working to rigorously characterize ApNGT mutants to expand the available repertoire of enzymes and develop more highly orthogonal NGT and GlycTag pairs. We note that at present the NGTs used in this work install a reducing end Glc rather than a GlcNAc. If a reducing end GlcNAc is required, previous work has shown that GlcN can be installed by ApNGT Q469A  and AaNGT and then further converted to GlcNAc by an acetyltranferase 13 . We are also working to engineer ApNGT Q469A  to directly install GlcNAc. 
     Conclusion 
     We have described the first systematic and generalizable method to site-specifically control glycosylation at multiple sites within the same protein, and that the development of enzymatic tools that can distinguish between chemically identical glycosylation sites described and demonstrated here overcomes the major limitation in achieving multiple, distinct, site-specifically defined glycoforms for basic science and biotechnological applications. 
     Methods 
     Phylogenetic analysis was performed using RaxmL, MUSCLE, and iTol. Peptide synthesis and SAMDI was performed similarly as Examples 1 and 2 and as described in the art 72 . Cell free protein synthesis was performed similarly as Examples 1 and 2 and as described in the art 72 . Peptide synthesis was performed using Wang-resin and purification was performed by HPLC. NGT purification was performed using a strept tag. The Im7 target was purified using a His tag. Modified protein and Glycoprotein with Glc, prepare for samples for LC-qTOF. 
     Special consideration for sequential modification of Im7 with NGTs including the following: To facilitate sequential purification steps, the Im7 sequence bearing four optimized GlycTags was fused to the C-terminus of an N-terminally polyhistidine-tagged SUMO protein. After reaction with HiNGT, the His-SUMO-Im7 protein was immobilized to magnetic beads then sequentially reacted with each subsequent NGT with wash steps in between NGT treatments. Finally, the Im7 was eluted and released from the SUMO fusion protein by cleavage with Ulp1 protease. 
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     In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. 
     All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. 
     Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.