Patent Publication Number: US-9896700-B2

Title: Engineered recombinant enzymes for methane oxidation

Description:
This invention was made with government support under grant number GM070473 AND awarded by the National Institutes of Health and 1346523 (STTR Grant) awarded by the National Science Foundation. The government has certain rights in the invention. 
    
    
     FIELD 
     Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided. 
     BACKGROUND 
     Methane, the primary component of natural gas, is a cheap and abundant feedstock that is costly to transport and requires significant capital expenditures to convert to higher value products (refs. 1, 2; incorporated by reference in their entireties). As a result, natural gas produced in remote locations such as the Bakken Shale is flared, leading to over $18 billion worth of methane being wasted per year (ref 3; incorporated by reference in its entirety). One potential solution is to use biological systems for methane conversion. These systems are predicted to require lower capital expenditures per barrel than traditional gas-to-liquid Technology (refs. 1, 2; incorporated by reference in their entireties). 
     In nature, methane is aerobically oxidized by bacteria known as methanotrophs, which utilize it for energy production and carbon fixation (ref. 4; incorporated by reference in its entirety). Methane enters the methanotroph metabolic pathway by the action of methane monooxygenases (MMOs), which oxidize methane to methanol (ref. 4; incorporated by reference in its entirety).
 
R—H+O 2 +2H + +2 e   − →R—OH+H 2 O
 
Nature employs two types of MMOs: soluble MMO (sMMO), which utilizes a diiron cofactor; and particulate MMO (pMMO), which utilizes a dicopper cofactor ( FIG. 1 ) (refs. 5, 6; incorporated by reference in their entireties). A major issue limiting the development of biological gas-to-liquid technology is the inability to express sMMO or pMMO in an industrially relevant host organism (refs. 1, 2; incorporated by reference in their entireties).
 
     SUMMARY 
     Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided. 
     In some embodiments, provided herein are compositions comprising a polypeptide capable of converting an alkane into an alkanol and/or an alkene to an epoxide. In some embodiments, the polypeptide is capable of converting methane into methanol. In some embodiments, the polypeptide is capable of converting propylene into propylene oxide. In some embodiments, the polypeptide carries out the hydroxylation of the alkane. In some embodiments, the polypeptide carries out the hydroxylation of the methane. In some embodiments, the polypeptide comprises a metal dependent oxygenase with an engineered active site. In some embodiments, the polypeptide is expressed in an industrially-relevant host organism and at a commercially-relevant scale. In some embodiments, the polypeptide comprises two domains, each with at least 60% sequence identity (e.g., &gt;60%, &gt;70%, &gt;75%, &gt;80%, &gt;90%, &gt;95%, &gt;98%, &gt;99%) with a domain of pmoB. In some embodiments, the polypeptide comprises S1-D1-linker-D2-S2, S1-D1-linker-D2, D1-linker-D2-S1, wherein S1 is a first soluble peptide, D1 is a polypeptide segment with at least 60% sequence identity (e.g., &gt;60%, &gt;70%, &gt;75%, &gt;80%, &gt;90%, &gt;95%, &gt;98%, &gt;99%) with a first soluble domain of pmoB, linker is a soluble peptide linker, D2 is a polypeptide segment with at least 60% sequence identity (e.g., &gt;60%, &gt;70%, &gt;75%, &gt;80%, &gt;90%, &gt;95%, &gt;98%, &gt;99%) with a second soluble domain of pmoB, and S2 is a second soluble peptide. In some embodiments, the polypeptide comprises at least 60% sequence identity (e.g., &gt;60%, &gt;70%, &gt;75%, &gt;80%, &gt;90%, &gt;95%, &gt;98%, &gt;99%) with spmoB7, sumo-spmoB7, and/or sumo-spmoB7 8pt3. 
     In some embodiments, provided herein are methods of converting an alkane (e.g., methane) into an alkanol (e.g., methanol) comprising exposing the alkane to a polypeptide described herein. 
     In some embodiments, provided herein are cells expressing a polypeptide described herein. 
     In some embodiments, provided herein are methods of biofuel production comprising exposing an alkane (e.g., methane) to a polypeptide described herein to produce an alkanol (e.g., methanol). 
     In some embodiments, provided herein are soluble polypeptides capable of converting an alkane into an alkanol comprising a core sequence comprising at least 60%, but less than 100% sequence identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, ranges there between) with SEQ ID NO: 23, the core sequence being C-terminally- and/or N-terminally-flanked by one or more soluble peptide segments. In some embodiments, the polypeptide is capable of converting methane into methanol. In some embodiments, the polypeptide carries out the hydroxylation of the alkane. In some embodiments, the polypeptide comprises a copper oxidase with an engineered active site. In some embodiments, the polypeptide is expressed in an industrially-relevant host organism and at a commercially-relevant scale. In some embodiments, the one or more soluble peptide segments are selected from peptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, ranges therein) sequence identity with one or SEQ ID NOS: 16, 17, 18, 21, and 22. In some embodiments, the soluble polypeptide comprises at least 60% sequence identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, ranges therein) with spmoB7 (SEQ ID NO: 13), sumo-spmoB7 (SEQ ID NO: 14), and/or sumo-spmoB7 8pt3 (SEQ ID NO: 13), but less than 100% sequence identify from a naturally-occurring sequence. 
     In some embodiments, provided herein are system comprising the soluble polypeptides capable of converting an alkane into an alkanol described herein. In some embodiments, systems comprise a fixed support selected from the list consisting of: a yeast cell, a phage, and a functionalized bead. 
     In some embodiments, provided herein are bioreactors comprising the soluble polypeptides capable of converting an alkane into an alkanol described herein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . (A) Crystal structure of sMMO (PDB code 4GAM). (B) Crystal structure of pMMO (PDB code 3RGB). Iron (A) and copper (B) ions are shown as spheres. 
         FIG. 2 . SpmoB7 activity assays and controls. 
         FIG. 3 . Sequence alignments of variants. 
         FIG. 4 . Improvements in soluble expression of N- and C-terminal fusions of spmoB. (A) N-terminal fusions. (B) C-terminal fusions. 
         FIG. 5 . Improvement in soluble expression of multi-domain fusions of spmoB. 
         FIG. 6 . Hydrophobic patches identified on spmoB by TRIAD using the SAP algorithm. Patch 1: V293, P294, F413, M414; Patch 2: I380, Y381, P383; Patch 3: P408, P411, I410; Patch 4: P280, L277, A279, P278; Patch 5: L109, P111; Patch 6: P169, V170; Patch 7: L175; Patch 8: P94, P96, G95. 
         FIG. 7 . Improvement in soluble expression of combinatorial mutants of spmoB. All of the combinatorial mutants shown here were built using D1-D2-set12, as in the library screening experiments. 
         FIG. 8 . Ancestry of variants by round of engineering. The starting template is spmoB. Combination mutants FC8pt1-4 are descended from a variant of spmoB with the Mc-6 linker, set12 C-terminal domain fusion, and M298F/M300C base double mutation. 
         FIG. 9 .  13 C methanol production by selected variants 
     
    
    
     DEFINITIONS 
     As used herein, the term “peptide” refers a short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are of about 50 amino acids or less in length. A peptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and/or modified amino acids. 
     As used herein, the term “polypeptide” refers a polymer of amino acids, linked together by peptide bonds, that is over about 50 amino acids or less in length. A polypeptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and/or modified amino acids. 
     As used herein, a “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 
     1) Alanine (A) and Glycine (G); 
     2) Aspartic acid (D) and Glutamic acid (E); 
     3) Asparagine (N) and Glutamine (Q); 
     4) Arginine (R) and Lysine (K); 
     5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); 
     6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W); 
     7) Serine (S) and Threonine (T); and 
     8) Cysteine (C) and Methionine (M). 
     Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (histidine (H), lysine (K), and arginine (R)); polar negative (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class. 
     In some embodiments, unless otherwise specified, a conservative or semi-conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and/or amino acid analogs. 
     Non-conservative substitutions (e.g., not conservative or semi-conservative) involve the exchange of an amino acid of one class or group for an amino acid from another class or group. 
     As used herein, the term “sequence identity” refers to the degree to which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) differ only by conservative (e.g., “conservative sequence similarity”) and/or semi-conservative (e.g., “semi-conservative sequence similarity”) amino acid substitutions. The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position. 
     As used herein, the term “physiological conditions” encompasses any conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, chemical makeup, etc. that are compatible with living cells. 
     The term “soluble”, particularly when used in reference to peptide, polypeptide or protein, as used herein refers to the characteristic of being substantially, completely dissolvable in aqueous solution, under, for example physiological conditions. For example, soluble polypeptide typically lacks any transmembrane segments. 
     As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Sample may also refer to cell lysates or purified forms of the peptides and/or polypeptides described herein. Cell lysates may include cells that have been lysed with a lysing agent or lysates such as rabbit reticulocyte or wheat germ lysates. Sample may also include cell-free expression systems. Such examples are not however to be construed as limiting the sample types applicable to the present invention. 
     As used herein, the term “bioreactor” refers to any vessel in which a chemical process or reaction (e.g., conversion of an alkane (e.g., methane) into an alkanol (e.g., methanol), etc.) is carried out which involves organisms or biochemically active substances (e.g., enzymes or polypeptides capable or advancing a chemical reaction without turnover). Process within a bioreactor may be aerobic or anaerobic. A bioreactor can be of any size so long as it is useful for the culturing of cells and/or the performance of the desired chemical reaction. Typically, the bioreactor will be at least 1 liter and may be 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. In some embodiments, the internal conditions of the bioreactor, including, but not limited to pressure, pH and temperature, are optionally controlled during the culture and/or reaction period. A bioreactor is composed of any material that is suitable for holding the bioreactive components (e.g., media, reactants, products, etc.) under appropriate conditions, such as glass, plastic or metal. A bioreactor may also comprise one or more ports, vents, valves, etc. for the additional and/or removal (e.g., selective additional and/or removal) or products, reactants, etc. 
     DETAILED DESCRIPTION 
     Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided. 
     pMMO is a membrane bound α3β3γ3 homotrimer with protomers of three polypeptides: pmoA, pmoB, and pmoC (ref. 7; incorporated by reference in its entirety). The active site of pMMO is a dicopper site that resides in the N-terminal soluble domain of pmoB (ref. 5; incorporated by reference in its entirety) pmoB contains two soluble domains, an N-terminal cupredoxin domain and a C-terminal cupredoxin domain, connected in the middle by two transmembrane helices (ref 7; incorporated by reference in its entirety). It has been demonstrated that the two-transmembrane helices of pmoB can be replaced with an artificial linker and expressed in  E. coli  (ref. 5; incorporated by reference in its entirety). This protein, commonly referred to as spmoB, expresses as insoluble protein that can be refolded in the presence of copper to obtain active protein. The refolded protein is relatively unstable and obtained in extraordinarily low yields (ref 5; incorporated by reference in its entirety). In some embodiments, a protein within the scope herein does not require in vitro refolding and/or in vitro metal loading. In some embodiments, protein within the scope herein is active inside of an industrially relevant host organism (e.g.,  E. coli ). 
     Using spmoB as a template, several computational protein design strategies were used to design in silico libraries to improve protein solubility. These strategies included: (1) designing the composition, length, and cutpoints for the linker between the two fragments of spmoB for improved stability and solubility, (2) stabilizing the hydrophobic core by identifying buried cavities and reducing their number and size, (3) redesigning the surface to reduce the number and size of hydrophobic patches, and (4) designing soluble fusion partners for N-terminus and C-terminus. Libraries encoding 2000 variants were screened for solubility and 5 variants with estimated expression yields of more than 10 mg/L in plasmid-based  E. coli -based expression systems were obtained. Variants with expression levels as high as 30 mg/L were obtained. Purification of a variant referred to as spmoB7 was carried out and activity assays indicate this variant oxidizes methane to methanol. Additional variants, sumo-spmoB7 and sumo-spmoB7 8pt3, were also shown to be active using isotopically-labeled methane. Reconstitution of the copper cofactor is carried out using an in vivo copper loading method (see Examples). spmoB7 is isolated from the soluble fraction of an  E. coli  lysate (no refolding necessary) and no additional modifications of the protein are needed to obtain activity. In some embodiments, the protein is in an active, functional, and/or folded state in vivo. In some embodiments, this variant is routinely isolated at expression levels above 5 mg/L (e.g., &gt;10 mg/L&gt;20 mg/L, &gt;30 mg/L, &gt;50 mg/L, or more). 
     Experiments conducted during development of embodiments of the present invention demonstrate that computational and screening methods used herein produced protein sequences resulting in active soluble fragments of pMMO, and indicate that other variants with sequences described below also oxidize methane (See Examples). 
     Embodiments described herein find use in a variety of applications, not limited to the following:
         Methanol production from methane—proteins described herein are used, for example, in conjunction with other biochemical pathways to enable utilization of methane via a methanol-dependent pathway for biofuel production in either cell-free systems or in vivo systems.   General oxidation chemistries—Since the chemistry performed by the protein described herein has not been previously demonstrated using recombinant proteins without in vitro refolding or metal loading, there are many alternative applications that stem from the construction of these polypeptides.       

     Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided. 
     In some embodiments, soluble engineered polypeptides comprise a first polypeptide having at least 60% sequence identity with a first soluble domain of pmoB linked (via a linker peptide) to a second polypeptide having at least 60% sequence identity with a first soluble domain of pmoB. In some embodiments, the first and second polypeptides are artificial sequences (e.g., not naturally-occurring) having less than 100% sequence identity with a naturally occurring pmoB sequence. 
     In some embodiments, soluble engineered polypeptides further comprise one or more soluble peptide sequences (e.g., sumo, set12, mbp, etc.) linked (directly or via a linker peptide) to the pmoB-like polypeptide domains. In some embodiments, a soluble peptide is attached to the C-terminus and/or N-terminus of the soluble engineered polypeptides. 
     In some embodiments, soluble engineered polypeptides comprise, a first soluble peptide portion (S1) (SEQ ID NOs: 16, 17, 18, 21, and/or 22; or variants thereof), two linked pmoB-like polypeptide domains (D1-linker-D2) (SEQ ID NO: 23; or variants thereof), and a second soluble peptide portion (S2) (SEQ ID NOs: 16, 17, 18, 21, and/or 22; or variants thereof). Polypeptides may comprise additional soluble peptides and/or linkers. In some embodiments, a soluble engineered polypeptide comprises S1-D1-linker-D2-52. 
     In some embodiments, soluble engineered polypeptides comprise a portion with at least 60% but less than 100% sequence identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) with SEQ ID NO: 23. In some embodiments, the polypeptides further comprise one or more soluble peptide sequences (e.g., sumo, set12, mbp, etc.) linked (directly or via a linker peptide) to the C-terminus and/or N-terminus of SEQ ID NO: 23. In some embodiments, the soluble peptide sequences comprise at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) sequence identity with one of SEQ ID NOs: 16, 17, 18, 21, or 22. 
     In some embodiments, soluble engineered polypeptides comprise a portion with at least 60% but less than 100% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence similarity (e.g., semi-conservative or conservative) with SEQ ID NO: 23. In some embodiments, the polypeptides further comprise one or more soluble peptide sequences (e.g., sumo, set12, mbp, etc.) linked (directly or via a linker peptide) to the C-terminus and/or N-terminus of SEQ ID NO: 23. In some embodiments, the soluble peptide sequences comprise at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence similarity (e.g., semi-conservative or conservative) with one of SEQ ID NOs: 16, 17, 18, 21, or 22. 
     In some embodiments, all or a portion of the polypeptides and peptide segments within the scope herein comprise at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence identity and/or similarity with one or more of SEQ ID NOS:10-23. In some embodiments, a soluble engineered polypeptide or a portion thereof comprises at least one substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, or ranges there between) relative to one of SEQ ID NOS:10-23. 
     EXAMPLES 
     Methane/Propylene Oxidation Assay Results 
     Experiments were conducted during development of embodiments of the present invention to demonstrate monooxygenase activity by detecting the oxidation of propylene to propylene oxide (PO). In MMO activity assays, PO is frequently detected instead of methanol because of PO&#39;s low background in typical biological solutions and its relative ease of detection (ref 5; incorporated by reference in its entirety). Table 1 shows that several variants were capable of oxidizing propylene. SpmoB7 was subsequently assayed for methane oxidation and was found to be capable of producing methanol as well ( FIGS. 2 and 9 , Table 2, and SEQUENCES). The methane oxidation measurements have considerable measurement error due to the difficulty of measuring methanol production at low levels. To overcome this obstacle and to conclusively show methanol is being produced from methane, activity of an spmoB7 variant with an N-terminally encoded sumo tag and a similar construct with additional point mutations were measured using isotopically labeled methane ( FIG. 9 ). The spmoB7, sumo-pmoB7, and sumo-spmoB7 8pt3 variants are the first examples of engineered fragments of pmoB that express solubly in  E. coli  and are active without refolding. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Summary of purified spmoB data collected during the granting period. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Soluble 
                 Propylene  
                 Methane 
               
               
                   
                 Cu ions  
                 expression  
                 oxidation  
                 oxidation  
               
               
                   
                 per 
                 yield 
                 activity 
                 activity 
               
               
                 Variant 
                 protein 
                 (mg/L) 
                 (min −1 ) 
                 (min −1 ) 
               
               
                   
               
               
                 parent spmoB 
                 5.4 
                 Negligible 
                 0.086 ± 0.037 
                  0.20 ± 0.020* 
               
               
                   
                 (n = 2) 
                   
                   
                   
               
               
                 spmoB2 
                 — 
                 11.4 
                 0.035 ± 0.0035 
                 — 
               
               
                 spmoB5 
                 2.5 ± 0.3  
                 32.1 
                 0.022 ± 0.0074 
                 — 
               
               
                   
                 (n = 2) 
                   
                   
                   
               
               
                 spmoB7 
                 0.7 ± 0.04 
                 17.3 
                 0.028 ± 0.014 
                 0.057 ± 0.051 
               
               
                   
                 (n = 3) 
                   
                   
                   
               
               
                 spmoB8 
                 — 
                 Negligible 
                 — 
                 — 
               
               
                 spmoB10 
                 — 
                 6.0 
                 — 
                 — 
               
               
                 spmoB12 
                 — 
                 15.1 
                 0.031 ± 0.029 
                 — 
               
               
                 sumo spmoB7 
                 — 
                 6.5 
                 — 
                 See FIG. 9 
               
               
                 sumo spmoB7 
                 — 
                 5.4 
                 — 
                 See FIG. 9 
               
               
                 8pt3 
               
               
                   
               
               
                 *Literature values reported under different conditions. 
               
            
           
         
       
     
                     TABLE 2                  Glossary of spmoB variant labels. The second column (“Nomenclature”)       defines the variant architecture using a common nomenclature        that is read from N-terminus to C-terminus, “D|1” and        “D2” correspond to domains 1 and 2, respectively, “Mc-6” and        “pmoA-gb1” are linker constructs that are defined in Table 3. “mbp,”       “set12,” and “sumo” are domain fusions; if a domain fusion label        appears on the left of “D1,” then it is an N-terminal fusion, and if on the        right of “D2,” a C-terminal fusion.                         Label   Nomenclature   Description/Notes               spmoB   D1-D2   pmoB subunit of  M. capsulatus                 (Bath) pMMO       Mc-6   D1-Mo-6-D2   spmoB with GEPSGEPS                linker (SEQ ID NO: 1)       pmoA-gb1   D1-pmoA-gb1-D2   spmoB with GEPS-gb1-               GE-pmoA-GS                linker (SEQ ID NO: 2)       set12   D1-Mc-6-D2-set12   Mc-6 with C- terminal fusion       spmoB2   D1-pmoA-gb1-D2-set12   pmoA-gb1- with                C-terminal fusion; measured                PO and MMO activity       spmoB5   mbp-D1-pmoA-   pmoA-gb1 with double fusion;            gb1-D2-set12   measured PO and MMO activity       spmoB7   set12-D1-pmoA-   pmoA-gb1 with double fusion;            gb1-D2-set12   mearured PO and MMO activity       spmoB8   set12-D1-pmoA-   pmoA-gb1 with double fusion;            gb1-D2-sumo   measured PO and MMO activity       spmoB10   mbp-D1-Mo-6-D2-sumo   Mc-6 with double fusion;                measured PO and MMO activity       spmoB12   set12-D1-Mc-6-D2-sumo   Mc-6 with double fusion;                measured PO and MMO activity       FC   D1-Mc6-D2-set12 +    set12 with M298F/M300C           M298F/M300C           sumo    set12-sumo-D1-pmoA-   spmoB7 with additional        spmoB7   gb1-D2-set12   N-terminal fusion                    
Generation of Models of Parent spmoB
 
     The original parent spmoB construct (ref. 5; incorporated by reference in its entirety), is derived from the pmoB subunit of  Methylococcus capsulatus  Bath (Mc) pMMO. The spmoB construct contains residues 22-172 and 256-414 of pmoB connected by a flexible Gly-Lys-Leu-Gly-Gly-Gly (GKLGGG) linker (SEQ ID NO: 3). Because no crystal structure of spmoB exists, a structural model for spmoB was generated using the loop modeling feature of TRIAD, Protabit&#39;s proprietary computational protein design software suite. The model was constructed by removing the two transmembrane helices (residues 172-265) from the coordinate file of pmoB derived from the crystal structure of holo-pMMO (PDB ID: 3RGB) (ref. 8; incorporated by reference in its entirety) and modeling the flexible linker into the resulting gap; TRIAD was used to find the lowest energy conformation and placement of the linker relative to the rest of spmoB. 
     Linker Designs for Improved Stability/Solubility 
     The initial linker inserted between the two domains of the parent spmoB is a six residue GKLGGG linker (SEQ ID NO: 3), which was designed by visual inspection of the holo-pMMO structure (ref. 5; incorporated by reference in its entirety). Detailed modeling of the spmoB linker region identified several opportunities for improvement. First, molecular dynamics (MD) analysis of the parent spmoB predicts that the linker is the most flexible part of the construct; a highly flexible linker may contribute to the low soluble expression of spmoB. To reduce flexibility, linkers with more complex sequences are used. Several higher-complexity sequences including Gly, Ser, Glu, and Pro amino acid types and also small proteins or protein fragments were tested (Table 3). Second, if the regions on either side of the linker are considered in addition to the linker itself, a flexible 14-residue tether connects the two structured regions of spmoB. In some embodiments, the flexibility of this region is reduced overall by decreasing the size of the tether. Thus, in addition to the cutpoints for the original linker at residues 172 and 265, a second set of cutpoints was selected at residues 169 and 267, reducing the length of the tether by 5 residues. 
     
       
         
           
               
             
               
                 TABLE3 
               
             
            
               
                   
               
               
                 Linkers designed and tested. 
               
            
           
           
               
               
               
               
               
            
               
                 Name 
                 Cutpoints 
                 Linker 
                 Length 
                 Type 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Parent spmoB 
                 173-265 
                 GKLGGG (SEQ ID NO: 3) 
                 6 
                 Original linker 
               
               
                   
               
               
                 Mc-1 
                 172-263 
                 GSGSG (SEQ ID NO: 4) 
                 5 
                 Gly/Ser 
               
               
                   
               
               
                 Mc-2 
                 172-263 
                 GSGSGS (SEQ ID NO: 5) 
                 6 
                 Gly/Ser 
               
               
                   
               
               
                 Mc-3 
                 172-263 
                 GSGSGSG (SEQ ID NO: 6) 
                 7 
                 Gly/Ser 
               
               
                   
               
               
                 Mc-4 
                 169-267 
                 GSGSGSG (SEQ ID NO: 6) 
                 7 
                 Gly/Ser 
               
               
                   
               
               
                 Mc-5 
                 169-267 
                 GSGSGSGS (SEQ ID NO: 7) 
                 8 
                 Gly/Ser 
               
               
                   
               
               
                 Mc-6 
                 169-267 
                 GEPSGEPS (SEQ ID NO: 1) 
                 8 
                 Gly/Ser/Glu/Pro 
               
               
                   
               
               
                 Mc-7 
                 169-267 
                 GSGEPSGS (SEQ ID NO: 8) 
                 8 
                 Gly/Ser/Glu/Pro 
               
               
                   
               
               
                 pmoA-gb1 
                 182-267 
                 GEPS-gb1-GE-pmoA-GS (SEQ 
                 96 
                 pmoA + domain 
               
               
                   
                   
                 ID NO: 2) 
                   
                   
               
               
                   
               
               
                 pmoA-b2m 
                 182-267 
                 GEPS-b2m-GE-pmoA-GS (SEQ 
                 139 
                 pmoA + domain 
               
               
                   
                   
                 ID NO: 2) 
                   
                   
               
               
                   
               
               
                 pmoA-b2mL 
                 182-267 
                 GEPS-b2mL-GE-pmoA-GS (SEQ 
                 189 
                 pmoA + domain 
               
               
                   
                   
                 ID NO: 2) 
               
               
                   
               
               
                 * the sequence of the pmoAlinker is PVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEK(SEQ ID NO: 9) 
               
            
           
         
       
     
     Several Gly/Ser and Gly/Ser/Glu/Pro linkers ranging in length from 4 to 11 residues were evaluated with TRIAD&#39;s loop modeling feature, which uses inverse kinematics (ref. 9; incorporated by reference in its entirety) and multiple rounds of relaxation to predict the most likely conformation and position of each linker relative to the rest of spmoB. Linkers containing less than 5 residues were found to be strained or not able to span the distance between the fixed cutpoints. Linkers of length 6 and higher did not show signs of strain and some were even predicted to form short secondary structure elements. 
     Seven short Gly/Ser and Gly/Ser/Glu/Pro linkers were constructed and evaluated for their effect on the soluble expression of spmoB (Table 2, Mc-1 to Mc-7). Soluble expression screening with the split-GFP assay (described below (ref 10; incorporated by reference in its entirety)) indicated that construct Mc-6 had the highest soluble expression of the group, with a marginal improvement in soluble expression over the parent. Further rounds of engineering were executed using Mc-6 as the background template. 
     A simple structure-based approach was used to recapitulate a more native-like linker between the two domains of spmoB. Using the crystallographic information from 3RGB and 3RFR (pMMO from  Methylocystis  sp. strain M) as a guide, a sequence from a soluble region of pmoA that interacts with pmoB was selected and incorporated as a natural linker between the two domains in the pmoB subunit. To achieve this, the cutpoint of pmoB was moved from residue 169 to 182, the construct was appended with a Gly-Glu-Pro-Ser linker, and an additional Gly-Ser linker was attached N-terminally to the cutpoint at residue 267. 
     The new linker and cutpoints create an empty 15 Å space from residue 182 of pmoB to where the pmoA subunit interacts with pmoB for 33 residues. Three different molecular entities were sampled at the 15 Å space: the soluble GB1 domain (ref 10; incorporated by reference in its entirety), beta-2-microglobulin (b2m) (ref 11; incorporated by reference in its entirety), and an extended b2m linker (ref 11; incorporated by reference in its entirety). The GB1 domain proved to be the most soluble of the three by the split-GFP assay on the spmoB Mc-6 template. This construct was named spmoB pmoA-gb1, and was later incorporated into downstream versions of the protein. 
     N- and C-Terminal Fusion Designs 
     A wide range of soluble protein domains were investigated for their effects on spmoB expression at both the N- and the C-termini. These domains included GB1 (ref 10; incorporated by reference in its entirety), b2m (ref. 11; incorporated by reference in its entirety), maltose binding protein (mbp), N-utilising substance A (NusA) (ref. 12; incorporated by reference in its entirety), small ubiquitin-like modifier (sumo) protein (ref. 12; incorporated by reference in its entirety), and two versions of highly charged peptides originating from the bacteriophage T7 minor capsid protein 10B (set6 and set12) (ref 12; incorporated by reference in its entirety). These domains were selected for their known performance as solubility-enhancing factors either from the literature (refs. 10-12; incorporated by reference in their entireties) or from in-house experiments. A split-GFP soluble expression assay showed that fusions containing GB1, sumo, mbp, and set12 yielded the best results overall ( FIG. 5 ), and fusions to the C-terminus were, in general, more effective than fusions to the N-terminus. 
     This first round of data informed a second round of domain fusion experiments, in which two fusion protein domains were attached to the parent spmoB protein, one at each terminus ( FIG. 6 ). Four double-fusion constructs were made, featuring either mbp or set12 at the N-terminus and set12 or sumo at the C-terminus. An additional four constructs were made with identical N- and C-terminal fusions, but with the Mc-6 linker replaced with the pmoA-gb1 linker described above. These combination constructs showed the further improved soluble expression of the spmoB variants ( FIG. 6 ), and six of these variants were selected for large-scale expression and further characterization. 
     Designs to Improve the Stability of spmoB 
     A strategy for improving the stability of spmoB was to optimize packing within the protein&#39;s core. Poorly packed residues were identified using three analysis methods in TRIAD. First, statistical residue energy (SRE) analysis, a proprietary multicomponent scoring function, was used to identify residues that are not compatible with their environment in the protein. This method also identifies sub-optimal residues on the protein&#39;s surface. Second, the interstitial space between the atoms in the solvent-excluded interior of the protein (solvent excluded volume) for each residue was compared to a statistical distribution of volumes from a database of thermophilic and mesophilic protein structures (ref. 13; incorporated by reference in its entirety). Third, positions where point mutations are predicted by molecular mechanics force fields to improve the stability of the protein were identified with a ΔΔG scan using FoldX (ref. 14; incorporated by reference in its entirety) and TRIAD ΔΔG with the Rosetta (ref. 16; incorporated by reference in its entirety) and Phoenix (ref. 17; incorporated by reference in its entirety) forcefields. 
     The residues identified by the three methods were probed by single or double site-saturation mutagenesis as shown in Table 4. Subsequently, combinatorial sequence designs were carried out to optimize regions surrounding poorly packed “seed” residues within the core of spmoB, which were identified based on a consensus of packing quality based on SRE and solvent excluded volume (Table 5). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Summary of spmoB single and double site stability libraries tested.  
               
               
                 Positions were identified by core packing, SRE, or ΔΔG analysis. 
               
            
           
           
               
               
               
               
            
               
                   
                 Degenerate 
                 Amino acid types 
                 Design  
               
               
                 Positions 
                 codon 
                 represented 
                 strategy 
               
               
                   
               
               
                 K36 
                 NNS 
                 all 
                 SRE 
               
               
                 E66 
                 NNS 
                 all 
                 ΔΔG 
               
               
                 G76 
                 NNS 
                 all 
                 SRE 
               
               
                 T80 
                 NNS 
                 all 
                 SRE/ΔΔG 
               
               
                 L89 
                 DKS 
                 R, C, G, I, L, M, F, S, W, V 
                 Core 
               
               
                 P96 
                 NNS 
                 all 
                 SRE 
               
               
                 G107 
                 NNS 
                 all 
                 SRE 
               
               
                 L109 
                 NNS 
                 all 
                 SRE 
               
               
                 D123 
                 NNS 
                 all 
                 ΔΔG 
               
               
                 D135 
                 NNS 
                 all 
                 SRE 
               
               
                 H137 
                 NNS 
                 all 
                 ΔΔG 
               
               
                 T140 
                 NNS 
                 all 
                 Core/ΔΔG 
               
               
                 N143 
                 NNS 
                 all 
                 ΔΔG 
               
               
                 Q145 
                 NNS 
                 all 
                 SRE 
               
               
                 G146 
                 NNS 
                 all 
                 SRE/ΔΔG 
               
               
                 G148 
                 NNS 
                 all 
                 SRE 
               
               
                 M163 
                 NNS 
                 all 
                 SRE 
               
               
                 T281 
                 NNS 
                 all 
                 ΔΔG 
               
               
                 D288 
                 NNS 
                 All 
                 SRE/ΔΔG 
               
               
                 M298 
                 DKS 
                 R, C, G, I, L, M, F, S, W, V 
                 Core 
               
               
                 M300 
                 DKS 
                 R, C, G, I, L, M, F, S, W, V 
                 Core 
               
               
                 N306 
                 NNS 
                 all 
                 ΔΔG 
               
               
                 S321 
                 NNS 
                 all 
                 ΔΔG 
               
               
                 I380 
                 NNS 
                 all 
                 SRE 
               
               
                 S385 
                 NNS 
                 all 
                 ΔΔG 
               
               
                 R400 
                 NNS 
                 All 
                 SRE 
               
               
                 D406 
                 NNS 
                 all 
                 SRE/ΔΔG 
               
               
                 L89/T140 
                 DKS/DBS 
                 R, C, G, I, L, M, F, S, W, V/A,  
                 Core 
               
               
                   
                   
                 R, C, G, I, L, M, F, S, T, W, V 
                   
               
               
                 M298/M300 
                 DKS/DKS 
                 R, C, G, I, L, M, F, S, W, V/R,  
                 Core 
               
               
                   
                   
                 C, G, I, L, M, F, S, W, V 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Seed positions and nearby residues 
               
               
                 included in combinatorial sequence design 
               
               
                 calculations. 
               
            
           
           
               
               
               
            
               
                   
                 Seed 
                 Nearby residues included in 
               
               
                   
                 residues 
                 design calculations| 
               
               
                   
                   
               
               
                   
                 L52 
                 W49, W54, F124, Y336 
               
               
                   
                 W54 
                 L52, I67, Y336 
               
               
                   
                 I67 
                 W54, V65, V126, L128, T140 
               
               
                   
                 L89 
                 W49, F124, V126, T140, M142 
               
               
                   
                 T140 
                 I67, L89, F124 
               
               
                   
                 M298 
                 A289, M300, A366, W371, L409 
               
               
                   
                 W371 
                 M298, F324, L376 
               
               
                   
                   
               
            
           
         
       
     
     For each seed position, the surrounding positions within 3 Å that interact with the sidechain of the seed position were chosen for design (Table 4). The design positions were allowed to retain their wild-type identities or mutate to any nonpolar amino acid identity. For all sequence design calculations, special care was taken to avoid designing positions that are known to coordinate the monocopper site (H48, H72) or the dicopper active site (H33, H137, and H139). 
     Using TRIAD, combinatorial sequence designs were carried out with variable design parameters, and the resulting sequences were converted into degenerate codon libraries with a target size of 250 variants. These variant sequences were also evaluated and ranked by solvent excluded volume to identify variants with improved packing quality. Sequence designs with seeds L89, M298, and W371 were chosen for further analysis because these designs yielded variants with the best solvent excluded volume. The resulting degenerate codon libraries are shown in Tables 6, 7, and 8. In the first round of engineering, testing focused on the single and double mutants in Table 4, which yielded several hits (see Table 9). The combinatorial sequence design libraries in Tables 6, 7, and 8 are evaluated in subsequent rounds of laboratory screening. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Degenerate codon library 1. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Degenerate 
               
               
                 Position 
                 Library AAs 
                 codon 
               
               
                   
               
               
                 M298 
                 A, I, L, M, P, T, V 
                 VYS 
               
               
                 F324 
                 R, Y 
                 TWC 
               
               
                 L376 
                 A, C, D, E, F, G, H, I, K, L, M,  
                 NBS + VNS 
               
               
                   
                 N, P, Q, R, S, T, V, W 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Degenerate codon library 2. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Degenerate 
               
               
                 Position  
                 Library AAs 
                 codon 
               
               
                   
               
               
                 W49 
                 F, W 
                 TTC + TGG 
               
               
                 L89 
                 I, M 
                 ATS 
               
               
                 V126 
                 F, I, L, M, V 
                 NTS 
               
               
                 T140 
                 I, M, V| 
                 RTS 
               
               
                 M142 
                 I, L, M, V 
                 VTS 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Degenerate codon library 3. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Degenerate 
               
               
                 Position  
                 Library AAs 
                 codon 
               
               
                   
               
               
                 M298 
                 F, I, M, V 
                 NTS 
               
               
                 M300 
                 F, I, L, M, V 
                 NTS 
               
               
                 W371 
                 C, F, L, R, W 
                 YKS 
               
               
                 L409 
                 L, M 
                 MTG 
               
               
                   
               
            
           
         
       
     
                                                     Library   #   #   #    Hit amino        Library   size   screened   sequenced   hits   acid identity                                                        K36   20   96   12   5   Q, V, P, E, M       E66   20   96   18   0   —       G76   20   96   34   2   R, E       T80   20   96   9   1   E       P96   20   96   8   2   G, A       G107   20   96   5   1   W       L109   20   96   4   1   D       D123   20   96   6   0   —       H137   20   96   5   2   E, D       T140   20   96   5   0   —       N143   20   96   23   2   G, S       G146   20   96   7   1   D       G148   20   96   13   1   L       M163   20   96   12   4   G, C, R, L       T281   20   96   4   0   —       D288   20   96   16   3   S, E, N       V293   18   96   21   1   A       S321   20   96   8   2   G, H       I380   20   96   4   0   —       S385   20   96   17   1   G       R400   20   96   6   0   —       D406   20   96   9   0   —       I410   18   96   8   0   —       L277/A279    324   768   28   1   L/P       M298/ M300   144   384   5   3   F/C, F/L, F/V       I380/Y381   324   768   38   6   A/E, E/S, G/S,                            T/D, T/G, G/D       F413/ M414   324   768   41   7   P/S, A/M, R/A, H/H,                             R/N, P/P, A/D                    
Designs to Remove Hydrophobic Patches on the Surface of spmoB
 
     To improve the solubility of spmoB, hydrophobic patches on the surface of spmoB were identified using the structure-based spatial aggregation propensity (SAP) algorithm (ref 17; incorporated by reference in its entirety). SAP calculations yielded eight hydrophobic patches as shown in  FIG. 6 . Many of the patches included prolines, which often serve important structural roles in proteins. Thus, proline positions were not mutated in any of the libraries. Three of the patches were excluded from consideration because of their location near or on the flexible linker that is being replaced in other variants (Patches 6 and 7), or because the patch is made up only of prolines and glycines, which often serve important structural roles and may be poor choices for design (Patch 8). 
     For each of the remaining patches, the degenerate codon VVW was chosen to replace the wild type residue at each position in the patch as shown in Table 10. This degenerate codon encodes 12 amino acids, which include all of the polar or charged amino acid types and was expected to improve the hydrophilicity at these positions on the surface of spmoB. Each position was mutated individually, resulting in 9 single site libraries; three combinations were also constructed for patches that have residues close in primary sequence (Table 10). L109 and 1380 were replaced with NNS instead of VVW because they were also identified by SRE as described above. Finally, residues F413 and M414 are located at the C-terminus of spmoB, which is exposed to the solvent. In addition to mutating these residues with the degenerate codon VVW, spmoB was truncated to remove either M414 or F413/M414. 
                     TABLE 10                  Summary of spmoB single and double site solubility        mutation libraries tested.                                     Degenerate   Amino acid types       Position   Patch #   codon   represented               L109*   5   NNS   all       L277   4   VVW   A, R, N, D, Q, E, G, H, K, P, S, T       A279   4   VVW   A, R, N, D, Q, E, G, H, K, P, S, T       V293   1   VVW   A, R, N, D, Q, E, G, H, K, P, S, T       I380*   2   NNS   all       Y381   2   VVW   A, R, N, D, Q, E, G, H, K, P, S, T       I410   3   VVW   A, R, N, D, Q, E, G, H, K, P, S, T       F413   1   VVW   A, R, N, D, Q, E, G, H, K, P, S, T       M414   1   VVW   A, R, N, D, Q, E, G, H, K, P, S, T       L277/A279   4   VVW/VVW   A, R, N, D, Q, E, G, H, K, P, S, T/                   A, R, N, D, Q, E, G, H, K, P, S, T       I380/Y381   2   VVW/VVW   A, R, N, D, Q, E, G, H, K, P, S, T       F413/M414   1   VVW/VVW   A, R, N, D, Q, E, G, H, K, P, S, T/                   A, R, N, D, Q, E, G, H, K, P, S, T               *indicates that position was also identified by SRE (Table 3)            
Construction and Cloning of Initial spmoB Gene
 
     The parent spmoB gene was cloned into pY71A(lc) using Gibson assembly (ref. 18; incorporated by reference in its entirety). The spmoB gene was cloned in frame with a C-terminal tag composed of a TEV cleavage site, (Gly3 Ser)2 linker, β-strand 11 of GFP (ref 19; incorporated by reference in its entirety), a Gly3 Ser linker and a Strep-tag (ref 20; incorporated by reference in its entirety). This arrangement of purification and split-GFP tags on the C-terminus was selected from a collection of tag arrangements that still exhibited the desired functionality of each tag. The combined tag was abbreviated to “TGS” and referenced as such within this document. 
     Construction of spmoB Variant Libraries 
     Variant libraries of spmoB were constructed using either the Q5® SDM Kit (NEB Inc., Ipswich, Mass., USA) or through megaprimer mutagenesis (ref. 21; incorporated by reference in its entirety). After transformation into BL21 Gold DE3 cells, colonies were picked on a Genetix Qbot into 384-well glycerol stock plates. Libraries were oversampled by at least 3-fold, and in most cases almost 5-fold. 
     Soluble Expression Screen Development 
     To identify spmoB variants with improved solubility, an in vitro split-GFP system was employed (ref. 19; incorporated by reference in its entirety). 96-well 2 mL deep well plates containing 1 mL of autoinduction media were inoculated with individual library colonies. Cultures were grown for 16 hours at 28° C. with shaking at 350 RPM. Plates were then centrifuged and the pellets were stored at −20° C. Cells were lysed using a detergent-based lysis buffer. Clarified lysate containing the expressed spmoB variants (with a C-terminal β-strand 11 from GFP) was mixed with the GFP1-10 reagent, allowing the full-length GFP protein to become reconstituted and fluoresce. The detected GFP fluorescence is proportional to the amount of soluble protein; therefore, a brighter signal indicates more soluble spmoB is present in the lysate. The advantages of this assay include: (1) the minimal effect of the C-terminal GFP11 tag on solubility of the protein of interest, ensuring the response is due only to the protein of interest, and (2) the selectivity of the split-GFP interaction, allowing the assay to be performed in clarified cell lysate. 
     The split-GFP complementation assay was automated with a Tecan Evo liquid-handling robot, allowing for 768 unique clones to be examined in triplicate each day. Assay plates had a set of control wells containing clones with the empty pY71A(lc) plasmid, the parent spmoB-C-set12 gene, or an unrelated solubly-expressed protein as the negative, baseline, and positive controls, respectively. Over the course of the project, the GFP1-10 reagent requirement was reduced 4-fold through the use of high density microplates (384-well vs. 96-well). The cost of the lysis buffer was also reduced by 66% while maintaining effectiveness by decreasing the amount of a commercial lytic additive and supplementing it with an off-the-shelf detergent. 
     Soluble Expression Screen Results 
     After split-GFP analysis, potential hits were re-arrayed, re-cultured, and sent to Beckman Genomics for single pass sequencing. True hits were identified by matching results from the sequencing analysis and data from a secondary split-GFP assay. Results are shown in Table 9. Library construction and solubility screening 
     Large Scale Growth, Purification, and Activity Assay of spmoB7 
     Eight constructs identified from domain fusion studies were selected for growth scale-up and purification (Table 1). Plasmids were transformed into  E. coli  strain C41(DE3) or BL21(DE3) and grown overnight on LB-agar plates containing ampicillin. Individual colonies were used to inoculate 1 L cultures of autoinduction media, then grown at 37° C. with shaking at 225 RPM. The growth temperature was changed to 20° C. at an OD600 of approximately 0.5. After 10 hours of growth, cells were transferred to 1 L centrifuge bottles along with 5 mL of sterile sugar solution and 5 mL of 1 M CuSO4. The sugar solution contains 125 g glycerol, 12.5 g glucose, and 50 g α-lactose monohydrate brought to a final volume of 500 mL with water. Resuspended cells were incubated for two hours at room temperature in sealed bottles. Cells were harvested by centrifugation and the cell pellet was flash frozen and stored at −80° C. 
     Frozen cell pellets were re-suspended in wash buffer (150 mM NaCl and 50 mM Tris base, pH 8.0) and lysed by sonication or chemical additives. Cellular debris was removed by ultracentrifugation at 40,000 RPM for 30 minutes. The resulting supernatant was applied to a streptactin column equilibrated with wash buffer using an FPLC. After extensive washing, the purified protein was eluted from the column using buffer containing 150 mM NaCl, 50 mM Tris base, 2.5 mM desthiolbiotin, and 10 mM MgCl2 at pH 8.0. Eluted protein was then analyzed using SDS-PAGE. The extinction coefficient, calculated by the program Geneious (Biomatters, Auckland, New Zealand) at 280 nm was used for all protein quantitation. Results indicate that four of the six constructs express at levels of &gt;10 mg/L, with one (spmoB5) expressing at &gt;30 mg/L (Table 1). 
     Metal Content Analysis of Hits 
     The activity of spmoB is contemplated to be dependent on both the correct coordination of copper ions within the active sites of the protein and the concentration of copper in the in vitro assay during methane oxidation; although the present invention is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention. The metal content of spmoB5 and spmoB7 were therefore determined using inductively coupled plasma-atomic emission spectroscopy (ICPAES). The average metal content of spmoB5 and spmoB7 shown in Table 1 is from 2 and 3 biological replicates (grown and purified independently), respectively. Subsequent activity assays indicate that there is not a direct correlation between metal content and activity. 
     Methane/Propylene Oxidation Assay Development 
     Duroquinol was prepared (22; incorporated by reference in its entirety) 20-100 μL of protein was placed in a gas chromatography (GC) vial with ˜1-2 mg of solid duroquinol and sealed. Typical protein concentrations were between 5 and 100 μM. 1 mL of propylene, methane or  13 C labeled methane was added using a syringe. Reactions were then incubated in a shaking water bath set at 20 to 45° C. After 1 to 24 hour(s), samples were moved to −20° C. for about 10 minutes. 500 μl of chloroform was added to samples containing propylene, shaken at 1800 RPM for 10 minutes, and centrifuged at 2,000×g for 2 minutes. The chloroform layer was then chromatographed with an Agilent 7890B gas chromatograph coupled to a 5977A MSD equipped with a 25 m×0.25 mm PorabondQ with particle traps. Single ion mode was used to quantitate the concentration of propylene oxide by monitoring the 58 m/z ion. Methylene chloride was used as an internal standard by monitoring the 49 m/z ion. 
     For samples containing methane, 500 μL of chloroform was added to each sample and shaken at 1800 RPM for 10 minutes. The samples were then centrifuged at 2,000×g for 2 minutes to clarify the emulsion. The chloroform layer was then transferred to a fresh vial and chromatographed as described above except that the 31 m/z ion and 33 m/z ion was used to quantitate the concentration of methanol and  13 C labeled methane. The rate of methane oxidation is calculated after correcting for background methanol. 
     Combination of Mutations from Top Performing Hits 
     The best single and double mutations obtained from the first round of design (K36Q/V, T80E, P96G/A, G146D/G148L, V293A, M298F/M300C, I380A/Y381E) were combined into the backbone of the screening construct D1-D2-set12. As shown in  FIG. 7 , these mutations all increase the solubility of the D1-D2-set12 construct to varying degrees and show a similar fold improvement when compared to the multi-domain fusions in  FIG. 5  and  FIG. 6 . The starting backbone was first mutated to include M298F/M300C, generating the FC construct, while the subsequent FC8pt mutations incorporated the following base mutations (T80E, G146D, G148L, V293A, I380A, Y381E), but differed in the combinations at K36Q/V and P96G/A (FC8pt1 K36Q/P96G, FC8pt2 K36Q/P96A, FC8pt3 K36V/P96G, FC8pt4 K36V/P96A). The best expressing variant based on the GFP assay was FC8pt3. Mutations in variant FC8pt3 were combined with sumo-SpmoB7 to generate sumo-SpmoB7 8pt3, which was capable of oxidizing  13 C labeled methane to  13 C methanol. 
     The number of positions considered presents numerous combinations for consideration when combining variants. Although all of the mutations in the FC8pt series are beneficial, we identified a smaller subset of mutations that provide similar levels of soluble expression. Additional variants include SS1 (K36V, P96G, G146D, M298F, M300C), SS2 (P96G, G146D, M298F, M300C) and the corresponding N-terminal sumo fusions. 
     Assessment of the Stability and Solubility of Best Variants 
     Variants of spmoB with increased solubility were expressed and purified to homogeneity using Streptactin resin followed by size exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare Life Science, Piscataway, N.J.). The purified proteins showed similar secondary structure to refolded spmoB when examined with circular dichroism; however, no discernible melting transition was observed for any of the variants. Thermofluor assays (ref. 23; incorporated by reference in its entirety) were also performed; however none of the constructs exhibited a thermal transition. It is contemplated that the spmoB proteins exist as a soluble aggregate, which can still allow for significant activity despite having non-traditional tertiary structure (ref. 24; incorporated by reference in its entirety); although the present invention is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention. 
     Soluble spmoB Variants in Eukaryotic Expression Systems 
     To test the transferability of the soluble spmoB protein variants, a sampling of early hits from the fusion domain studies were cloned into a  Pichia pastoris -compatible vector (ref. 25; incorporated by reference in its entirety) The variants were cloned in frame with the alpha mating factor secretion signal, ensuring secretion of the protein out of the cell and into the media. Despite potential difficulties not accounted for during solubility engineering, such as glycosylation, measurable amounts of protein was recovered for one of the variants tested. This shows for the first time the expression of the spmoB protein solubly by a eukaryotic microbe and secreted into the medium. 
     Similarly, soluble spmoB variants were cloned into the  Saccharomyces cerevisiae  plasmid pPNL6, in frame with the yeast surface protein aga2 to promote yeast display (ref. 26; incorporated by reference in its entirety). Under control of the galactose promoter, spmoB variants were successfully displayed on the surface of yeast as measured by flow cytometry. Roughly 40% of cells counted were fluorescent in response to labeling with an anti-cmyc primary antibody and an anti-IgG secondary antibody conjugated to phycoerythrin. 
     
       
         
           
               
             
               
                   
               
               
                 SEQUENCES 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 In some embodiments, sequences include a C-terminal TGS tag. 
               
               
                 spmoB-C-set12 (SEQ ID NO: 10) 
               
               
                 MHGEKSQAAFMRMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFL 
               
               
                 NVGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQG 
               
               
                 GGPIIGPGKWITVEGSMSEFRNGEPSGEPSGTMRGMKPLELPAPTVSVKVEDATYRVPG 
               
               
                 RAMRMKLTITNHGNSPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAEDGLSVSDNSPL 
               
               
                 APGETRTVDVTASDAAWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQVVQIDAPLIPSFM 
               
               
                 ENLYFQGGEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQGSGGGSTSRDH 
               
               
                 MVLHEYVNAAGITGGGSAWSHPQFEK 
               
               
                   
               
               
                 spmoB2 (D1-pmoA-gb1-D2-set12)(SEQ ID NO: 11) 
               
               
                 MHGEKSQAAFMRMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFL 
               
               
                 NVGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQG 
               
               
                 GGPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDLENGEPSTYKLILNGKTLKGETTTEAV 
               
               
                 DAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGEPVEYNGMLMSIADIQGYNYV 
               
               
                 RTGTPEYIRMVEKGSGTMRGMKPLELPAPTVSVKVEDATYRVPGRAMRMKLTITNHGN 
               
               
                 SPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAEDGLSVSDNSPLAPGETRTVDVTASDA 
               
               
                 AWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQVVQIDAPLIPSFMENLYFQGGEEASVTS 
               
               
                 TEETLTPAQEAAETEAANKARKEAELEAETAEQGSGGGSTSRDHMVLHEYVNAAGITG 
               
               
                 GGSAWSHPQFEK 
               
               
                   
               
               
                 spmoB5 (mbp-D1-pmoA-gb1-D2-set12)(SEQ ID NO: 12) 
               
               
                 MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGP 
               
               
                 DIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY 
               
               
                 NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGK 
               
               
                 YDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAW 
               
               
                 SNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEA 
               
               
                 VNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINA 
               
               
                 ASGRQTVDEALKDAQTNENLYFQGHGEKSQAAFMRMRTIHWYDLSWSKEKVKINETV 
               
               
                 EIKGKFHVFEGWPETVDEPDVAFLNVGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFR 
               
               
                 VVLKARRPGDWHVHTMMNVQGGGPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDLENG 
               
               
                 EPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE 
               
               
                 GEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEKGSGTMRGMKPLELPAPTVSVKVE 
               
               
                 DATYRVPGRAMRMKLTITNHGNSPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAEDGL 
               
               
                 SVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQVVQID 
               
               
                 APLIPSFMENLYFQGGEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQGSGG 
               
               
                 GSTSRDHMVLHEYVNAAGITGGGSAWSHPQFEK 
               
               
                   
               
               
                 spmoB7 (set12-D1-pmoA-gb1-D2-set12)(SEQ ID NO: 13) 
               
               
                 MEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQENLYFQGHGEKSQAAFM 
               
               
                 RMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFLNVGMPGPVFIRK 
               
               
                 ESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQGGGPIIGPGKWITVE 
               
               
                 GSMSEFRNPVTTLTGQTVDLENGEPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYA 
               
               
                 NDNGVDGEWTYDDATKTFTVTEGEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEK 
               
               
                 GSGTMRGMKPLELPAPTVSVKVEDATYRVPGRAMIRMKLTITNHGNSPIRLGEFYTASV 
               
               
                 RFLDSDVYKDTTGYPEDLLAEDGLSVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIIY 
               
               
                 DPDSRFAGLLFFFDATGNRQVVQIDAPLIPSFMENLYFQGGEEASVTSTEETLTPAQEAA 
               
               
                 ETEAANKARKEAELEAETAEQGSGGGSTSRDHMVLHEYVNAAGITGGGSAWSHPQFEK 
               
               
                   
               
               
                 sumo-spmoB7 (set12-sumo-D1-pmoA-gb1-D2-set12)(SEQ ID NO: 14) 
               
               
                 MEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQENLYFQGLQDSEVNQEA 
               
               
                 KPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGI 
               
               
                 RIQADQAPEDLDMEDNDBEAHREQIGGHGEKSQAAFMRMRTIHWYDLSWSKEKVKIN 
               
               
                 ETVEIKGKFHVFEGWPETVDEPDVAFLNVGMPGPVFIRKESYIGGQLVPRSVRLEIGKTY 
               
               
                 DFRVVLKARRPGDWHVHTMMNVQGGGPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDL 
               
               
                 ENGEPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFT 
               
               
                 VTEGEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEKGSGTMRGMKPLELPAPTVSV 
               
               
                 KVEDATYRVPGRAMRMKLTITNHGNSPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAE 
               
               
                 DGLSVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQV 
               
               
                 VQIDAPLIPSFMENLYFQGGEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQ 
               
               
                 GSGGGSTSRDHMVLHEYVNAAGITGGGSAWSHPQFEK 
               
               
                   
               
               
                 sumo-spmoB7 FC8pt3 (SEQ ID NO: 15) 
               
               
                 MEIASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQENLYFQGLQDSEVNQEA 
               
               
                 KPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRKLMEAFAKRQGKEMDSLRFLYDGI 
               
               
                 RIQADQAPEDLDMEDNDIIEAHREQIGGHGEVSQAAFMRMRTIHWYDLSWSKEKVKIN 
               
               
                 ETVEIKGKFHVFEGWPEEVDEPDVAFLNVGMPGGVFIRKESYIGGQLVPRSVRLEIGKTY 
               
               
                 DFRVVLKARRPGDWHVHTMMNVQDGLPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDL 
               
               
                 ENGEPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFT 
               
               
                 VTEGEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEKGSGTMRGMKPLELPAPTVSV 
               
               
                 KVEDATYRAPGRAFRCKLTITNHGNSPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAE 
               
               
                 DGLSVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIAEDPDSRFAGLLFFTDATGNRQV 
               
               
                 VQIDAPLIPSFMENLYFQGGEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQ 
               
               
                 GSGGGSTSRDHMVLHEYVNAAGITGGGSNWSHPFEK 
               
               
                   
               
               
                 set12 (SEQ ID NO: 16) 
               
               
                 EEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQ 
               
               
                   
               
               
                 ENLYFQG (SEQ ID NO: 17) 
               
               
                 ENLYFQG 
               
               
                   
               
               
                 sumo (SEQ ID NO: 18) 
               
               
                 LQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEM 
               
               
                 DSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG 
               
               
                   
               
               
                 pmoA-gb1-D2 (SEQ ID NO: 19) 
               
               
                 PVTTLTGQTVDLENGEPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDG 
               
               
                 EWTYDDATKTFTVTEGEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEKGSGTMRG 
               
               
                 MKPLELPAPTVSVKVEDATYRVPGRAMRMKLTITNHGNSPIRLGEFYTASVRFLDSDVY 
               
               
                 KDTTGYPEDLLAEDGLSVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIIYDPDSRFAG 
               
               
                 LLFFFDATGNRQVVQIDAPLIPSFMENLYFQGG 
               
               
                   
               
               
                 D1 (SEQ ID NO: 20) 
               
               
                 HGEKSQAAFMRMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFLN 
               
               
                 VGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQGG 
               
               
                 GPIIGPGKWITVEGSMSEFRN 
               
               
                   
               
               
                 TGS(SEQ ID NO: 21) 
               
               
                 GSGGGSTSRDHMVLHEYVNAAGITGGGSAWSHPQFEK 
               
               
                   
               
               
                 Mbp (SEQ ID NO: 22) 
               
               
                 MKIEEGKLVIWINGDKGYNGLQSGLLAEITPDKAKDTGIKVTVEHPDKLEEKFPQVAAT 
               
               
                 GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEA 
               
               
                 LSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKY 
               
               
                 ENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGP 
               
               
                 WAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDE 
               
               
                 GLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA 
               
               
                 VINAASGRQTVDEALKDAQTNENLYFQG 
               
               
                   
               
               
                 D1-pmoA-gb1-D2 (SEQ ID NO: 23) 
               
               
                 HGEKSQAAFMRMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFLN 
               
               
                 VGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQGG 
               
               
                 GPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDLENGEPSTYKLILNGKTLKGETTTEAVD 
               
               
                 AATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGEPVEYNGMLMSIADIQGYNYVR 
               
               
                 TGTPEYIRMVEKGSGTMRGMKPLELPAPTVSVKVEDATYRVPGRAMRMKLTITNHGNS 
               
               
                 PIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAEDGLSVSDNSPLAPGETRTVDVTASDA 
               
               
                 AWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQVVQIDAPLIPSFMENLYFQGG 
               
               
                   
               
            
           
         
       
     
     REFERENCES 
     The following references, many of which are referenced above by number, are herein incorporated by reference in their entireties.
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