METHOD FOR ULTRA-RAPIDLY SELECTING SIGNAL PEPTIDE TO WHICH INDIVIDUAL BARCODE SYSTEM FOR INCREASING PROTEIN PRODUCTIVITY IS INTRODUCED

The present invention relates to a composition for screening various signal peptides to select specific ones that allow efficient secretion of a target protein to out of host cells. The present invention also relates to a method for selecting specific signal peptides that express a target protein in host cells and efficiently secrete the target protein to out of the host cells. The use of the composition and/or method according to the present invention enables ultrafast selection of optimal signal peptides for a target protein through barcoding sequences corresponding to the signal peptides, leading to the maximization of the production yield of the recombinant protein.

TECHNICAL FIELD

The present invention relates to a composition including barcoding sequences for screening various signal peptides to select specific ones that allow efficient secretion of a to target protein to out of host cells.

BACKGROUND ART

With recent advances in biotechnology, a great deal of research around the world has focused on the structure and function of proteins. The explosive growth of the market for recombinant protein pharmaceuticals, including therapeutic antibodies, that are specific to target diseases and have few side effects is shaking up the global pharmaceutical market that has been dominated by small-molecule drugs.

Recombinant protein drugs refer to therapeutic proteins that are mass-produced in microbial or animal cell systems using genetic recombination technology. Therapeutic proteins have previously been difficult to obtain in vivo. Recombinant proteins can be modified such that they are expressed intracellularly or secreted extracellularly. When overexpressed intracellularly, recombinant proteins often accumulate into inactive insoluble aggregates in cells. Although recombinant proteins do not accumulate, there may arise many factors negatively affecting productivity, such as cumbersome cell disruption and difficult isolation and purification from numerous other proteins present in cells. These negative factors can be easily avoided by extracellular secretion of produced proteins. Extracellular secretion of proteins requires correct folding and modification of proteins after transcription, enabling the production of soluble proteins that are activated and have accurate tertiary structures for therapeutic protein production. Thus, the production of recombinant proteins via extracellular secretion is advantageous in terms of protein production yield as well as protein quality control. Therefore, optimization for efficient extracellular secretion of recombinant proteins is important in the manufacture of recombinant protein drugs.

Signal peptides are amino acid sequences that are located at the N-termini of secretory or membrane proteins and function as targeting signals for the secretory or membrane proteins. Very diverse signal peptides are known so far. For example, approximately 4,000-5,000 signal peptides are found in eukaryotes. The extracellular secretion rate of a target protein may vary depending on which signal peptides are applied for the production of the target protein. In this connection, several studies have been reported aimed at selecting signal peptides matched to target proteins and optimizing the extracellular secretion of the target proteins (Acta Biochim Biophys Sin(Shanghai) (2011) 43 (2): 96-102, Signal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteins.Biochem Biophys Res Commun.2010 Jan. 1; 391(1): 931-5, Signal peptide design for improving recombinant protein secretion in the baculovirus expression vector system).

In conclusion, ultrafast screening of currently known 4,000 or more signal peptides to select the most suitable ones for the expression of target proteins and introduction of the selected signal peptides into the target proteins in the development of protein drugs can artificially induce the maximum production of recombinant protein drugs.

The description of the Background Art is merely provided for better understanding of the background of the invention and should not be taken as corresponding to the prior art already known to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

Problems to be Solved by the Invention

The present inventors have earnestly and intensively conducted research to find a method for rapidly searching the most suitable signal peptides that allow highly efficient extracellular secretion of a target protein. As a result, the present inventors have found that when specific amino acids are combined into barcoding sequences corresponding to signal peptides, a library of the barcoding sequences and the signal peptides is constructed and expressed in host cells, and the amounts of a target protein in culture media of the host cells are determined, optimal signal peptides for efficient extracellular secretion of the target protein can be selected. Based on this finding, the present invention has been accomplished.

Therefore, it is one object of the present invention to provide a composition for screening various signal peptides to select specific ones that allow efficient secretion of a target protein to out of host cells.

It is a further object of the present invention to provide a method for selecting specific signal peptides that allow secretion of a target protein to out of host cells after expression of the target protein in the host cells.

Other objects and advantages of the invention become more apparent from the following detailed description, claims, and drawings.

Means for Solving the Problems

One aspect of the present invention provides a composition for screening various signal peptides to select specific ones that allow efficient secretion of a target protein to out of host cells.

The present inventors have made efforts to find a method for rapidly searching for the most suitable signal peptides that allow highly efficient extracellular secretion of a target protein. As a result, the present inventors have found that when specific amino acids are combined into barcoding sequences corresponding to signal peptides and the barcoding sequences are applied a target protein, optimal signal peptides for efficient extracellular secretion of the target protein can be selected.

As used herein, the term “signal peptide” refers to a peptide that is fused to the N-terminus (front end) of a secretory protein secreted from cells and allows the protein to pass through the cell membrane. The signal peptide is usually composed of 10 to 30 amino acids and is cleaved off by a specific transmembrane protease. The signal peptide allows only the secretory protein to secrete extracellularly. A total of 4137 signal peptides are known so far to be present in eukaryotic cells (Uniprot, June, 2017).

Extracellularly secreted proteins have their own signal peptides. That is, secreted proteins are expected to have different signal peptides depending on the type of expressing cells, the role of the proteins after expression or the expression of appropriate amounts of the proteins.

As used herein, the term “target protein” refers to a protein that is to be produced in proper host cells with high efficiency. The introduction of optimal signal peptides for a target protein into the recombinant protein in the development of a biopharmaceutical can artificially induce the maximum production of the target protein.

According to a preferred embodiment of the present invention, the composition includes polypeptides containing signal peptide tags (SP-tags) or nucleic acid molecules encoding the polypeptides.

As used herein, the term “signal peptide tag” or “SP-tag” refers to a tag that contains a barcoding sequence site consisting of three or more amino acids barcoding the corresponding signal peptide. The signal peptide tag can be fused to the C-terminus (rear end) of the target protein either directly or via a linker.

According to a preferred embodiment of the present invention, the three or more amino acids are selected from the group consisting of leucine (L), proline (P), alanine (A), tryptophan (W), tyrosine (Y), threonine (T), serine (S), glutamate (E), and aspartate (D).

Three out of the nine amino acids may be combined into a codon. In this case, a total of 729 (9×9×9) signal peptides can be barcoded with the codons. Alternatively, five out of the nine amino acids may be combined into a codon. In this case, a total of 59,049 (9×9×9×9×9) signal peptides can be barcoded with the codons. Considering that 4,137 signal peptides have been discovered so far in eukaryotes, each of the barcoding sequences preferably consists of 3 to 8 amino acids, more preferably 3 to 5 amino acids.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently consists of the same three amino acids selected from the nine amino acids.

For example, each of the barcoding sequences may be independently selected from the group consisting of LLL, PPP, AAA, WWW, YYY, TTT, SSS, EEE, and DDD.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently consists of the same two amino acids and one different amino acid selected from the nine amino acids.

For example, each of the barcoding sequences may independently have a configuration in which two identical amino acids are repeated consecutively, such as LLP, PPA, AAW, WWY, YYT, TTS, SSE, EED or DDL, or in which one different amino acid is inserted between two identical amino acids, such as LDL, PEP, ASA, WTW, YLY, TPT, SAS, EWE or DYD.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently consists of three different amino acids selected from the nine amino acids.

For example, the barcoding sequence may be selected from LPA, PAW, AWY, WYT, YTS, TSE, SED, EDL, and DLP, each of which consists of a series of different amino acids.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently consists of four different amino acids selected from the nine amino acids.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently consists of a combination of four amino acids selected from the nine amino acids wherein some of the four amino acids are identical and some of the four amino acids are different.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently consists of five identical amino acids selected from the nine amino acids.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently consists of a combination of five amino acids selected from the nine amino acids wherein some of the five amino acids are identical and some of the five amino acids are different.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently includes or is independently the sequence set forth in SEQ ID NO: 1:

with the proviso that [L]1, [L]2or [L]3is optionally replaced with an amino acid selected from the group consisting of P, A, W, Y, T, S, E, and D.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently includes or is independently the sequence set forth in SEQ ID NO: 2:

with the proviso that [L]1, [L]2, [L]3or [L]4is optionally replaced with an amino acid selected from the group consisting of P, A, W, Y, T, S, E, and D.

According to a preferred embodiment of the present invention, each of the barcoding sequences independently includes or is independently the sequence set forth in SEQ ID NO: 3:

with the proviso that [L]1, [L]2, [L]3, [L]4or [L]5is optionally replaced with an amino acid selected from the group consisting of P, A, W, Y, T, S, E, and D.

In the Examples section that follows, the nine amino acids were selected as constituents for the barcoding sequences as follows:

(1) First, 18 amino acids were selected. K and R as trypsin cleavage sites were excluded from the 20 amino acids, which will be described later.

(2) 12 out of the 18 amino acids were selected. Specifically, V, F, G, C, H, and N with high or low hydropathy indices were excluded from the 18 amino acids. First, the expression levels of peptides, each containing a different one of the 18 amino acids, were measured by LC-MS analysis. As a result, the amino acids V, F, G, C, H, and N were found to have relatively high or low hydropathy indices (particularly, H is likely to affect His tags and change its charge).

(3) M was excluded from the 12 amino acids. M is susceptible to oxidation modification during protein digestion. A total of 11 amino acids were selected.

(4) I and Q were excluded from the amino acids I, Q, N, L, E, and D with similar masses (N was already excluded in (2)) and L, E and D only were used. Finally, the nine selected amino acids L, P, A, W, Y, T, S, E, and D were used to design the barcoding sequences.

According to a preferred embodiment of the present invention, each of the signal peptide tags may independently further include a proteolytic cleavage site at the N-terminus (front end) of the barcoding sequence such that the barcoding sequence is cleaved by the protein.

As used herein, the term “proteolytic cleavage site” refers to a site that is present in a protein or peptide to be cleaved and is recognizable by a specific protein, resulting in cleavage of the target protein or peptide.

Various proteolytic cleavage sites are known in the art. Examples of preferred proteolytic cleavage sites include, but are not limited to, trypsin cleavage sites, thrombin cleavage sites, enterokinase cleavage sites, Factor Xa cleavage sites, collagenase cleavage sites, and TEV protease cleavage sites.

According to a preferred embodiment of the present invention, the proteolytic cleavage site is a trypsin cleavage site.

Trypsin is a proteolytic enzyme that cleaves the peptide chain next to lysine (K) or arginine (R). The proteolytic cleavage site may be selected from the group consisting of K and R.

The proteolytic cleavage site may be directly linked to the barcoding sequence. Alternatively, the proteolytic cleavage site may be linked to the barcoding sequence via a linker. The linker may be any of those known in the art but is preferably glycine (G).

In the Examples section that follows, glycine (G) was selected as the linker. This selection is based on the following considerations. The presence of P next to K causes incomplete trypsin cleavage and the acidic residues D and E next to K slow down hydrolysis rate, resulting in miscleavage. Accordingly, P, D, and E are unsuitable as linkers. L, P, A, W, Y, T, S, E, and D used in the barcoding sequences are excluded for stable cleavage.

According to a preferred embodiment of the present invention, the linker includes at least one glycine (G).

According to a preferred embodiment of the present invention, each of the polypeptides containing signal peptide tags independently further includes an affinity tag at the end of the barcoding sequence. The affinity tag is used to isolate and purify the barcoding sequence from culture media of the host cells.

As used herein, the term “affinity tag” refers to a tag that binds to a molecule of interest (e.g., a barcoding sequence) to allow isolation and purification of the molecule of interest using an affinity tag receptor. Various affinity tags known in the art can be used to implement the present invention.

The affinity tag may be selected from the group consisting of histidine tag (His-tag), myc-tag, FLAG-tag, small ubiquitin-like modifier tag (SUMO-tag), covalent yet dissociable NorpD peptide tag (CYD-tag), heavy chain of protein C tag (HPC-tag), calmodulin binding peptide tag (CBP-tag), and hemagglutinin-tag (HA-tag). The affinity tag is preferably a His-tag composed of 2 to 15 histidine residues.

Each of the signal peptide tags may be independently represented by Structure 1:

Each of the signal peptide tags may be independently represented by Structure 2:

More preferably, each of the signal peptide tags is independently represented by Structure 3:

The introduction of arginine (R) as a trypsin cleavage site at the C-terminus of the peptide ensures ionization stability of the peptide.

In the Examples section that follows, when a His tag was used as the affinity tag, arginine (R) was added to the C-terminus to ensure ionization stability because the terminal H is likely to change its charge. Arginine (R) was used also when isotope-labeled peptides were synthesized as internal standards to ensure quantitation. For reference, the arginine residue was15N- and13C-labeled.

According to a preferred embodiment of the present invention, the target protein is fused to the N-termini (front ends) of the signal peptide tags of the polypeptides.

According to a preferred embodiment of the present invention, the signal peptides are fused to the N-terminus (front end) of the target protein.

A further aspect of the present invention provides a vector including each of the nucleic acid molecules encoding the polypeptides containing signal peptide tags.

The nucleic acid molecule may be an isolated or recombinant one. Examples of such nucleic acid molecules include single- and double-stranded DNA and RNA and their corresponding complementary sequences. The isolated nucleic acid may be isolated from a naturally occurring source. In this case, the isolated nucleic acid is separated from the peripheral gene sequence present in the genome of a subject from which the nucleic acid is to be isolated. The isolated nucleic acid may be a nucleic acid, for example, a PCR product, a cDNA molecule or an oligonucleotide, that is enzymatically or chemically synthesized from a template. In this case, the nucleic acid produced from this procedure can be understood as the isolated nucleic acid molecule. The isolated nucleic acid molecule represents a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is “operably linked” when arranged in a functional relationship with another nucleic acid sequence. For example, the DNA of a presequence or secretory leader is operably linked to the DNA of the polypeptide when expressed as a preprotein, which is a presecretory polypeptide. A promoter or an enhancer affecting the transcription of the polypeptide sequence is operably linked to a coding sequence or a ribosome-binding site is operably linked to a coding sequence when it is arranged such that translation is promoted. Generally, the term “operably linked” means that DNA sequences to be linked are located adjacent to each other. In the case of secretory leaders, the term “operably linked” means that the secretory leaders are present adjacent to each other in the same leading frame. However, an enhancer needs not be contiguous. The linkage is performed by ligation at a convenient restriction enzyme site. In the case where this site does not exist, a synthetic oligonucleotide adaptor or a linker is used according to a suitable method known in the art.

As used herein, the term “vector” is used to refer to a carrier into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence may be “exogenous,” or “heterologous”. Examples of such vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophage). One of skill in the art may construct a vector through standard recombinant techniques (Maniatis et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In:Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

As used herein, the term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of regulatory sequences. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

As described above, a nucleic acid sequence encoding a polypeptide containing a signal peptide tag including a barcoding sequence encoded with specific amino acid codes corresponding to a signal peptide is constructed to have a “signal peptide-target protein-signal peptide tag” structure. The nucleic acid sequence can be inserted into a vector and expressed in a suitable host cell. In the case where the signal peptide allows efficient secretion of the “target protein-signal peptide tag” to the cell membrane of the host cell, the type of the signal peptide allowing efficient secretion of the target protein can be readily determined by validating the barcoding sequence of the signal peptide tag.

Another aspect of the present invention provides a method for selecting specific signal peptides that express a target protein in host cells and secrete the target protein to out of the host cells, the method including:

1) constructing vectors for various signal peptides to establish a library;

2) transforming host cells with the vectors;

3) expressing polypeptides containing signal peptide tags from the transformed host cells; and

4) quantifying the polypeptides containing signal peptide tags, the signal peptide tags or barcoding sequences of the signal peptide tags secreted to out of the transformed host cells.

As used herein, the term “host cell” refers to any transgenic organism that is capable of replicating the vector or expressing the gene encoded by the vector. Suitable organisms include eukaryotes and prokaryotes.

As used herein, the term “transformation” is intended to include “transfection” and “transduction”. The host cell may be transfected, transduced or transformed with the vector. This process means the delivery or introduction of the exogenous nucleic acid molecule into the host cell.

According to a preferred embodiment of the present invention, the method may further include 3-1) isolating and purifying the polypeptides containing signal peptide tags using affinity tags after step 3).

The method may further include 3-2) treating the polypeptides containing signal peptide tags with trypsin after step 3). The trypsin cleavage enables efficient isolation of the target protein and the barcoding sequences.

Steps 3-1) and 3-2) may be carried out individually or sequentially or in the reverse order after step 3).

In step 4), the polypeptides containing signal peptide tags, the signal peptide tags or the barcoding sequences of the signal peptide tags may be quantified by any suitable technique known in the art. Examples of such quantification techniques include, but are not limited to, protein chip analysis, immunoassay, ligand binding assay, matrix desorption/ionization time of flight (MALDI-TOF) mass spectrometry, surface enhanced laser desorption/ionization time of flight (SELDI-TOF) mass spectrometry, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical analysis, immunocytochemical analysis, immunoprecipitation, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LCMS), liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), Western blot, and enzyme linked immunosorbent assay (ELISA).

In the Examples section that follows, the results of quantification of the polypeptides containing signal peptide tags, the signal peptide tags or the barcoding sequences of the signal peptide tags by Western blot were compared with the results of ultrafast quantification of the polypeptides containing signal peptide tags, the signal peptide tags or the barcoding sequences of the signal peptide tags by LC-MS/MS. The ultrafast quantification of the barcoding sequences by LC-MS/MS revealed that three of the signal peptides had the highest expression levels. This result was consistent with the result of quantification by Western blot, which validated the efficacy of ultrafast quantification by LC-MS/MS.

For ease of ultrafast peptide quantification by LC-MS/MS, it is preferable that the signal peptide tags are up to 20 amino acids in length. It is more preferable that in Structure 1, each of the linkers consists of 1 to 3 amino acids, the barcoding sequence consists of 3 to 5 amino acids, and the affinity tag consists of 2 to 10 amino acids such that the full length is up to 20 amino acids. This construction is suitable for quantification by LC-MS/MS.

Effects of the Invention

The features and advantages of the present invention are summarized as follows:

(i) The composition of the present invention is effective in screening various signal peptides to select optimal signal peptides for efficient extracellular secretion of a target protein.

(ii) The method of the present invention is effective in selecting specific signal peptides that express a target protein in host cells and secrete the target protein to out of the host cells.

(iii) The use of the composition and/or method according to the present invention enables ultrafast selection of optimal signal peptides for a target protein through barcoding sequences corresponding to the signal peptides, leading to the maximization of the production yield of the recombinant protein.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be more specifically explained with reference to the following examples. It will be evident to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

EXAMPLES

Experimental Materials and Methods

1. Selection of Amino Acids for Use in Barcoding Sequences

For efficient and equivalent ionization and detection in LC-MS/MS, 18 amino acids were selected and used in barcoding sequences. K and R as trypsin cleavage sites were excluded from a total of 20 amino acids. Tags, each containing a different one of the 18 amino acids, were fused to the C-terminus of one target protein. As a result, a total of 18 clones were constructed.

A target gene was constructed by overlapping PCR (SET15-R500, Solgent). The vectors and the gene were treated with restriction enzymes AscI (R0558S, New England Biolabs)/XhoI (R0146S, New England Biolabs). The vectors and the PCR products were mixed with a DNA ligation mixture (6023, Takara), ligated at room temperature for 10 min, and transformed into DH5α (CP010, Enzynomics) for cloning. 18 plasmids were expressed in FREESTYLE™ 293-F cells (R79007, ThermoFisher Scientific) and the cell culture media were harvested 3 days later. The target protein in the cell culture media was isolated and purified by His affinity chromatography on Ni-NTA resin (05893801001, Roche). After pretreatment with the 18 proteins (each 1 μg), peak area values were calculated by LC-MS/MS quantification and were compared.

2. Signal Peptide and SP-Tag Vector Construction

Primers for 1200 SP-tags were synthesized (Cosmogenetech, Republic of Korea) and SP-tag genes were constructed by overlapping PCR (SET15-R500, Solgent). The vectors and the PCR products were treated with restriction enzymes AscI (R0558S, New England Biolabs)/XhoI (R0146S, New England Biolabs), followed by DNA ligation (6023, Takara). The ligation mixture was transformed into DH5α (CP010, Enzynomics) for cloning.

3. Validation of Protocol for Target Protein Library Construction

10 vectors carrying 10 signal peptides and one target protein gene were separately treated with restriction enzymes AscI (R0558S, New England Biolabs)/XhoI (R0146S, New England Biolabs). The 10 vectors and the inserts were subjected to DNA ligation (6023, Takara). The ligation mixture was transformed into DH5α (CP010, Enzynomics) to construct a mini-library. To demonstrate the diversity of the mini-library, re-transformation was performed and 100 colonies were picked and sequenced (Solgent, Republic of Korea).

4. Validation of Signal Peptide Screening

Vectors carrying 9 SP-tags and one target protein gene were separately cloned to construct 9 clones. The 9 clones were mixed together or independently expressed in FREESTYLE™ 293-F cells (R79007, ThermoFisher Scientific). 3 days later, cell culture media were harvested. Western blot was performed using anti-6× His antibody (ab18184, Abcam) to detect the target protein in the cell culture media. After pretreatment of the cell culture media by His affinity chromatography on Ni-NTA resin (05893801001, Roche), LC-MS/MS was performed to obtain peak area values. The Western blot results and LC-MS/MS results were analyzed and compared.

5. Quantitation by Multiple Reaction Monitoring (MRM)

MRM was performed on a nano LC system (nanoACQUITY UPLC™ System Nano-LC, Waters) connected to a hybrid triple quadruple/ion trap mass spectrometer (6500QTRAP, AB SCIEX, USA) with a nanoelectrospray interface. The mass spectrometer was operated in positive ion MRM mode in which Q1 and Q3 were set to transmit different precursor/product ion pairs.

The LC buffer system was as follows: mobile phase A, 2% acetonitrile/0.1% formic acid (Fisher Scientific, Cat #LS118-4) and mobile phase B, 98% acetonitrile/0.1% formic acid (Fisher Scientific, Cat #LS120-212). The peptides were separated and eluted at a flow rate of 300 nl/min on a linear concentration gradient of mobile phase B from 5% to 95% in 45 min. The total LC run time was 60 min. A trap (100 Å, 5 μm, 180 μm×20 mm, 2G, V/M, Cat #186006527) and an analytical column (130 Å, 1.7 μm, 100 μm×100, Cat #186003546) were used.

Typical instrument settings were as follows: ion spray (IS) voltage 2.3 kV, interface heater temperature 300° C., (nebulizer gas) setting 15, and curtain gas setting 20. MS coefficients for declustering potential (DP) and collision energy (CE) were determined by linear regression of optimized values for corresponding peptides and manual tuning. MRM experiments were conducted with a scan time of 20 ms and a scan width of 0.002 m/z using resolutions of 0.2 and 0.7 Da (FWHM) for Q1 and Q3, respectively. In the MRM runs, the scan time was maintained at 20 ms for each transition and the pause between transition scans was set to 1 ms. MRM transitions were selected to monitor fragment ions with the highest intensity.

Proteins were treated for MRM analysis as follows. Sample proteins were quantified by bicinchoninic acid (PIERCE™ BCA Protein Assay Kit, THERMO SCIENTIFIC™, 23225) assay. The protein samples (each 100 μg) were denatured with 6 M urea (GE HEALTHCARE, PN 17-1319-01), 50 mM Tris, pH 8.0 (Invitrogen, AM9855G), and 30 mM dithiothreitol (DTT, Sigma-Aldrich, PN D0632-10G) at 37° C. for 60 min and alkylated with 50 mM iodoacetamide (IAA, Sigma-Aldrich, PN I1149-25G) at room temperature in the dark for 30 min. Urea was diluted 15-fold with 50 mM Tris (pH 8.0), trypsin/Lys-C (Promega, PN V0571) was added in a 1:50 (w/w) enzyme-to-protein concentration ratio, followed by culture at 37° C. overnight.

Tryptic digestion was stopped by the addition of formic acid (SIGMA Aldrich, F0507) at a final concentration of 1% and desalting was performed on OASIS cartridges (Waters, PN WAT094225). The cartridges were equilibrated with 3 ml of water containing 0.1% formic acid and 3 ml of methanol before use. After use, the cartridges were washed with 3 ml of 0.1% formic acid and eluted with 1 ml of 60% ACN and 0.1% formic acid. The eluted samples were dried on a speed vacuum and frozen. Prior to MRM analysis, the samples were dissolved to a concentration of 0.2 μg/μl in 0.1% formic acid.

Each SP-tag consists of a cleavage site, a barcoding sequence, and a His tag. At least 14-20 amino acids can be used in SP-tags. Up to 20 amino acids can be quantified by LC-MS analysis. The present inventors utilized 14 amino acids in the following manner. SP-tags without quantitative differences were designed from 1200 peptides with different masses while maintaining their length at 14 amino acids.

(1) Barcoding Sequence Selection

(1) Protein peptides containing at least 8 amino acids and up to 20 amino acids were used for MRM analysis. R or K as a trypsin cleavage site was excluded from the peptides. That is, 18 out of the 20 amino acids were selected.

(2) Then, 12 out of the 18 amino acids were selected. Specifically, V, F, G, C, H, and N with high or low hydropathy indices were excluded from the 18 amino acids. First, the expression levels of peptides, each containing a different one of the 18 amino acids, were measured by LC-MS analysis to determine whether they were the same or different. As a result, the amino acids V, F, G, C, H, and N were found to have relatively high or low hydropathy indices (Particularly, H is likely to affect His tags and change its charge).

(3) M was excluded from the 12 amino acids. M is susceptible to oxidation modification during protein digestion. A total of 11 amino acids were selected.

(4) I and Q were excluded from the amino acids I, Q, N, L, E, and D with similar masses (N was already excluded in (2)) and L, E and D only were used. Finally, the nine selected amino acids L, P, A, W, Y, T, S, E, and D were used to design barcoding sequences consisting of 5 amino acids.

(2) Cleavage Site and His Tag Design

For trypsin cleavage, lysine (K) as a cleavage site was introduced in front of the peptide. For peptide ionization stability and quantification, arginine (R) was introduced at the end of the peptide.

G was introduced after the lysine (K) to prevent the appearance of continuous sequences such as KP, KD, and KE. The reasons for the selection of G were as follows: (1) P, D, and E were excluded because the presence of P next to K causes incomplete trypsin cleavage and the acidic residues D and E next to K slow down hydrolysis rate, resulting in miscleavage; (2) L, P, A, W, Y, T, S, E, and D were excluded because they were used in the barcoding sequences; and (3) G was suitable for stable cleavage.

6× histidine tags were attached for selective isolation and purification of the target to protein.

Experimental Results

1. Design of Method Using Signal Peptide Tags (SP-Tags) to Find Corresponding Signal Peptides

Signal peptides at the N-terminus of a protein that is translated in cells play a very important role in protein secretion. Grafting of various signal peptides for recombinant protein production promotes extracellular protein secretion, leading to an increase in production yield. The present inventors have succeeded in developing an ultrafast search method to find optimal signal peptides for protein production. Since signal peptides at the N-terminus of a protein are cleaved in cells, the signal peptides are not present in the secreted protein. Thus, the present inventors have found that when signal peptide tags (SP-tags) are attached to the C-terminus of a protein and are identified, the corresponding signal peptides can be determined (FIG. 1).

FIG. 2schematically shows a method for screening signal peptides capable of increasing protein production using SP-tags. Individual steps of the method will be explained below.

1) Target protein library construction: A target protein is cloned into vectors carrying various signal peptides and corresponding SP-tags with different molecular weights to construct a target protein library.

2) Expression of the target protein library: Several days after transfection of the target protein library into animal cells, cell culture media containing the protein secreted to out of the cells are collected. The amount of the secreted protein varies depending on the type of the signal peptide and can be determined by the SP-tag corresponding to the signal peptide.

3) Selection of signal peptides capable of increasing protein production: The SP-tags in the cell culture media containing the produced protein are identified and their relative amounts are quantified by LC-MS/MS. The identified SP-tags are ranked according to their relative amounts and the corresponding signal peptides are determined to find optimal ones capable of increasing protein production.

2. Construction of Library of Signal Peptides and SP-Tags

The target protein between the signal peptides and the SP-tags was cloned using restriction enzymes ASC I-Xba I/Nhe I to construct vectors having signal peptide-target protein-SP-tag structures (FIG. 3).

As shown inFIG. 4, vectors carrying 1200 signal peptides were cloned with 1200 SP-tags corresponding to the signal peptides to construct a library. The 1200 signal peptides used for library construction were signal peptides of already known human secretory proteins (Table 1).

3. Design of SP-Tags Capable of Distinguishing 1200 Signal Peptides

SP-tags were designed to consist essentially of barcoding sequences and histidine tags that produced 1200 or more diversities (FIG. 5). The barcoding sequences were designed to consist of 5 amino acids so as to correspond to 1200 signal peptides one by one. For the accuracy of subsequent experiments, 6× Histidine tags were attached to isolate and purify only the target protein from the cell culture media. Lysine (K) and arginine (R) were introduced at both ends for effective trypsin cleavage when LC-MS/MS was performed. Glycine residues as linker amino acids were inserted between the protein and the barcoding sequences and between the barcoding sequences and the His tags.

A total of 18 amino acids other than K and R as trypsin cleavage sites were selected as amino acids for the barcoding sequences. 18 SP-tagged proteins, each of which contains a different one of the 18 amino acids, were expressed and purified (FIG. 6).

Equal amounts of the 18 proteins were determined by LC-MS/MS to select amino acids suitable for relative quantification (FIG. 7). As a result, 6 (V, F, G, C, H, and N) out of the 18 amino acids were found to be unsuitable for quantification due to their relatively high or low values. The other 12 amino acids (I, L, P, A, W, Y, P, T, S, E, Q, and D) did not affect quantification due to their similar peak area values. That is, the 12 amino acids were judged to be suitable for relative quantification because their peak area values were determined only by their amounts irrespective of their types. As a result, the 12 amino acids were primarily selected for barcoding sequence design.

4. Design of Barcoding Sequences Consisting of Five Amino Acids to Construct Library of 1200 SP-Tags with Different Molecular Weights

For effective discrimination on LC-MS/MS, 1200 SP-tags are required to have different molecular weights. Hence, barcoding sequences were designed such that all sequences differed by ≥2 in molecular weight. Among the 12 selected amino acids, I and L, E and Q, and D and N were identical to or different from each other (within ˜±1) in molecular weight. Thus, I, Q, and N were excluded and only L, E, and D were used. Barcoding sequences consisting of five amino acids selected from L, P, A, W, Y, T, S, E, and D were designed to construct a library (Table 2).

5. Determination of Method for Effective Target Library Construction

Since it was impossible to clone a library of 1200 vectors at one time, a method for effective target library construction was tested. One target protein gene was cloned into vectors carrying 10 signal peptides (Table 3). A ligation-transformation-plasmid prep was conducted on a mixture of the same amounts of 10 vectors. In order to investigate the distribution of the signal peptides in the plasmids, each of the plasmids was prepped from 100 colonies and the corresponding signal peptides were sequenced (Table 4).

6. Validation of Method for Screening Signal Peptides Capable of Increasing Recombinant Protein Production

The effectiveness of a method for quantification of proteins expressed in a library by LC-MS/MS was verified by comparison with a method for quantification of independently expressed proteins by Western blot (FIG. 9). Several days after 9 signal peptide/SP-tag clones introduced with target proteins were mixed and transfected into animal cells, the SP-tagged proteins secreted to the cell culture media were relatively quantified by LC-MS/MS and ranked according to their relative amounts. Simultaneously, several days after 9 SP-tagged vectors were independently transfected in 9 different flasks, the expression levels of the SP-tagged proteins secreted to the cell culture media were measured by Western blot and compared (FIG. 10).

The resulting LC-MS/MS peaks obtained based on the library expression revealed that Constructs Nos. 8, 1, and 5 showed the highest expression levels in this order and Constructs Nos. 3 and 6 followed in this order (FIGS. 10 to 12). Constructs Nos. 2, 4, 7, and 9 could not be ranked because their expression levels were significantly low. The results of Western blot for the independently expressed constructs showed that Constructs Nos. 1, 8, and 5 were ranked as the top 3 constructs because of their highest expression levels and Constructs Nos. 7, 6, 9, 4, 3, and 2 followed in this order (FIGS. 10 and 11). In light of the aim of the present invention to select signal peptides that can best increase the production of a target protein, the same top 3 constructs determined in both experiments verified the effectiveness of the method for screening signal peptides using the SP-tag library.

7. Test for Screening Optimal Signal Peptides Using SP-Tags

Human vascular endothelial growth factor (hVEGF) as a target protein was cloned into the signal peptide/SP-tag library to construct a target protein library. Three days after expression of the library in HEK 293 cells, the cell culture media were collected and the expressed protein was isolated and purified using His tags. After treatment of the protein samples with trypsin, the peak area values were calculated by LC-MS/MS to determine the relative amounts of the SP-tags (FIG. 13).

The resulting LC-MS/MS peaks revealed that there were distinct differences in peak height, sharpness, and noise intensity depending on whether the expression levels were high or low (FIG. 14). SP-tags with high expression levels appeared as clearly distinguishable single peaks, whereas SP-tags with low expression levels were not distinguished from noise signals. Signal peptides corresponding to the top 10 SP-tags whose expression levels were found to be high inFIG. 13were selected (FIG. 15).

8. Synthesis of Standard Peptides for SP-Tag Quantification

To develop an MRM assay for screening 1,200 signal peptides, isotope labeled peptides (containing15N and13C-labeled arginine residues) based on 1,200 barcoding sequences were synthesized as internal standards (JPT Peptide Technologies, Berlin, Germany). Each of the 1,200 internal standards was added at a concentration of 100 fmol/μl to the protein expression mixture prior to MRM analysis. The internal standards were used to accurately distinguish between non-specific peaks and target peaks based on their retention times (RT) during MRM analysis. A global standard peptide (EQVTNVGGAVVTGVTAVAQK) was synthesized based on an isotope-labeled peptide (containing15N and13C-labeled lysine residues) to minimize errors between groups during analysis. The global standard peptide had a peptide sequence that was absent present in the samples. The global standard peptide was added at a concentration of 50 fmol/μl to the protein expression mixture prior to MRM analysis.

TABLE 5List of chemically synthesized SP tag PeptidesNumberSequenceNumberSequenceNumberSequenceNumberSequencePeptide_001GAAPAPGHHHHHHRPeptide_301GSYTLSGHHHHHHRPeptide_601GWWPAAGHHHHHHRPeptide_901GEYPWPGHHHHHHRPeptide_002GTAPAAGHHHHHHRPeptide_302GSLSYTGHHHHHHRPeptide_602GAAPWWGHHHHHHRPeptide_902GLLPYWGHHHHHHRPeptide_003GALSAAGHHHHHHRPeptide_303GTWPAPGHHHHHHRPeptide_603GSYTYPGHHHHHHRPeptide_903GWWDADGHHHHHHRPeptide_004GAASLAGHHHHHHRPeptide_304GTAPWPGHHHHHHRPeptide_604GYYTLAGHHHHHHRPeptide_904GEWTWAGHHHHHHRPeptide_005GSADAAGHHHHHHRPeptide_305GELTLPGHHHHHHRPeptide_605GYATYLGHHHHHHRPeptide_905GWAEWTGHHHHHHRPeptide_006GAATATGHHHHHHRPeptide_306GDLDLPGHHHHHHRPeptide_606GEWPAEGHHHHHHRPeptide_906GEATWWGHHHHHHRPeptide_007GTASASGHHHHHHRPeptide_307GTLELPGHHHHHHRPeptide_607GPAEWEGHHHHHHRPeptide_907GDADWWGHHHHHHRPentide_008GSASATGHHHHHHRPeptide_308GEWPAAGHHHHHHRPeptide_608GALELWGHHHHHHRPeptide_908GTAEWWGHHHHHHRPeptide_009GPAPASGHHHHHHRPeptide_309GWAPAEGHHHHHHRPeptide_609GLLSLWGHHHHHHRPeptide_909GYYSYPGHHHHHHRPeptide_010GSAPAPGHHHHHHRPeptide_310GPAEWAGHHHHHHRPeptide_610GEYSAYGHHHHHHRPeptide_910GPWDLYGHHHHHHRPeptide_111GALPAAGHHHHHHRPeptide_311GAAEWPGHHHHHHRPeptide_611GSAEYYGHHHHHHRPeptide_911GPYDWLGHHHHHHRPentide_012GAAPALGHHHHHHRPeptide_312GWLSAPGHHHHHHRPeptide_612GTADYPGHHHHHHRPeptide_912GPLDYWGHHHHHHRPeptide_013GSATAPGHHHHHHRPeptide_313GSWPALGHHHHHHRPeptide_613GELELEGHHHHHHRPeptide_913GYYDAYGHHHHHHRPeptide_014GALTAAGHHHHHHRPeptide_314GSAPWLGHHHHHHRPeptide_614GYYSLSGHHHHHHRPeptide_914GYADYYGHHHHHHRPeptide_015GAATLAGHHHHHHRPeptide_315GYYSAAGHHHHHHRPeptide_615GSYSLYGHHHHHHRPeptide_915GDWDYPGHHHHHHRPeptide_016GEASAAGHHHHHHRPeptide_316GYASAYGHHHHHHRPeptide_616GEWDLAGHHHHHHRPeptide_916GPWDYDGHHHHHHRPeptide_017GTADAAGHHHHHHRPeptide_317GELELAGHHHHHHRPeptide_617GDWELAGHHHHHHRPeptide_917GWYTLLGHHHHHHRPeptide_018GSASLAGHHHHHHRPeptide_318GEAELLGHHHHHHRPeptide_618GEADWLGHHHHHHRPeptide_918GYYSYTGHHHHHHRPeptide_019GAASLSGHHHHHHRPeptide_319GALELEGHHHHHHRPeptide_619GWYPAPGHHHHHHRPeptide_919GEWEYAGHHHHHHRPeptide_020GDASASGHHHHHHRPeptide_320GDLTLLGHHHHHHRPeptide_620GYAPWPGHHHHHHRPeptide_920GEYEWAGHHHHHHRPeptide_021GSATATGHHHHHHRPeptide_321GTLDLLGHHHHHHRPeptide_621GSWDLLGHHHHHHRPeptide_921GWAEYEGHHHHHHRPeptide_022GTAPAPGHHHHHHRPeptide_322GSWPADGHHHHHHRPeptide_622GLLTWTGHHHHHHRPeptide_922GYAEWEGHHHHHHRPeptide_023GAAPAEGHHHHHHRPeptide_323GSAPWDGHHHHHHRPeptide_623GTLTLWGHHHHHHRPeptide_923GEAEYWGHHHHHHRPeptide_024GPLSAAGHHHHHHRPeptide_324GTAPWTGHHHHHHRPeptide_624GWATAWGHHHHHHRPeptide_924GWYSLEGHHHHHHRPeptide_025GSLPAAGHHHHHHRPeptide_325GWLDAAGHHHHHHRPeptide_625GAATWWGHHHHHHRPeptide_925GSWEYLGHHHHHHRPeptide_026GPASLAGHHHHHHRPeptide_326GWADALGHHHHHHRPeptide_626GTYSYTGHHHHHHRPeptide_926GSYEWLGHHHHHHRPeptide_027GSADAPGHHHHHHRPeptide_327GAADWLGHHHHHHRPeptide_627GSYTYTGHHHHHHRPeptide_927GEKSWYGHHHHHHRPeptide_028GTATAPGHHHHHHRPeptide_328GDLSLEGHHHHHHRPeptide_628GYLELPGHHHHHHRPeptide_928GDWTYLGHHHHHHRPeptide_029GALDAAGHHHHHHRPeptide_329GELTLPGHHHHHHRPeptide_629GPYELLGHHHHHHRPeptide_929GDYTWLGHHHHHHRPeptide_030GSATLAGHHHHHHRPeptide_330GPLSYPGHHHHHHRPeptide_630GEWTAEGHHHHHHRPeptide_930GDLTWYGHHHHHHRPeptide_031GAASLTGHHHHHHRPeptide_331GLLPAYGHHHHHHRPeptide_631GEADWDGHHHHHHRPentide_931GEWSYDGHHHHHHRPeptide_032GDATASGHHHHHHRPeptide_332GEATWAGHHHHHHRPeptide_632GDAEWDGHHHHHHRPeptide_932GSYEWDGHHHHHHRPeptide_033GTASADGHHHHHHRPeptide_333GAADWDGHHHHHHRPeptide_633GDWDLSGHHHHHHRPeptide_933GEYTWTGHHHHHHRPeptide_034GSLSASGHHHHHHRPeptide_334GWLSATGHHHHHHRPeptide_634GTLSWEGHHHHHHRPeptide_934GTYTWEGHHHHHHRPeptide_035GDAPAPGHHHHHHRPeptide_335GTWSLAGHHHHHHRPeptide_635GWWSASGHHHHHHRPentide_935GWYPAYGHHHHHHRPeptide_036GTLPAAGHHHHHHRPeptide_336GWASLTGHHHHHHRPeptide_636GSASWWGHHHHHHRPeptide_936GYYPAWGHHHHHHRPeptide_037GTAPALGHHHHHHRPeptide_337GTLSAWGHHHHHHRPeptide_637GEYDLPGHHHHHHRPeptide_937GWAPYYGHHHHHHRPeptide_038GAATLPGHHHHHHRPeptide_338GSATWIGHHHHHHRPeptide_638GELDYPGHHHHHHRPeptide_938GYAPYWGHHHHHHRPeptide_039GEASAPGHHHHHHRPeptide_339GPLDAYGHHHHHHRPeptide_639GWYTAPGHHHHHHRPeptide_939GPWDWPGHHHHHHRPeptide_040GTAPADGHHHHHHRPeptide_340GEWSASGHHHHHHRPeptide_640GTWPAYGHHHHHHRPeptide_940GELDYYGHHHHHHRPeptide_041GSASLPGHHHHHHRPeptide_341GSAEWSGHHHHHHRPeptide_641GWATYPGHHHHHHRPeptide_941GDLEYYGHHHHHHRPeptide_042GALSLAGHHHHHHRPeptide_342GTWSADGHHHHHHRPeptide_642GYATWPGHHHHHHRPeptide_942GWWTLPGHHHHHHRPeptide_043GEADAAGHHHHHHRPeptide_343GTATWTGHHHHHHRPeptide_643GTAPYWGHHHHHHRPeptide_943GWLPWTGHHHHHHRPeptide_044GDLSAAGHHHHHHRPeptide_344GSWSLSGHHHHHHRPeptide_644GEYTLLGHHHHHHRPeptide_944GTWPWLGHHHHHHRPeptide_045GSADALGHHHHHHRPeptide_345GPADYDGHHHHHHRPeptide_645GELTLYGHHHHHHRPeptide_945GTLPWWGHHHHHHRPeptide_046GTATLAGHHHHHHRPeptide_346GEYTAPGHHHHHHRPeptide_646GTLEYLGHHHHHHRPeptide_946GWYTAYGHHHHHHRPeptide_047GAATLTGHHHHHHRPeptide_347GEAPYPGHHHHHHRPeptide_647GYAEWAGHHHHHHRPeptide_947GYYTAWGHHHHHHRPeptide_048GEASATGHHHHHHRPeptide_348GTYSLPGHHHHHHRPeptide_648GAAEYWGHHHHHHRPeptide_948GEYDYDGHHHHHHRPeptide_049GSADADGHHHHHHRPeptide_349GSYTLPGHHHHHHRPeptide_649GWYSALGHHHHHHRPeptide_949GDYEYDGHHHHHHRPeptide_050GDATATGHHHHHHRPeptide_350GALTYLGHHHHHHRPeptide_650GWLSYAGHHHHHHRPeptide_950GTYEYEGHHHHHHRPeptide_051GTLSASGHHHHHHRPeptide_351GEAEYAGHHHHHHRPeptide_651GYLSAWGHHHHHHRPeptide_951GWWELAGHHHHHHRPeptide_052GTASLSGHHHHHHRPeptide_352GAAEYEGHHHHHHRPeptide_652GEAEYEGHHHHHHRPeptide_952GWLEWAGHHHHHHRPeptide_053GAASAYGHHHHHHRPeptide_353GSYELAGHHHHHHRPeptide_653GEYSLEGHHHHHHRPeptide_953GWAEWLGHHHHHHRPeptide_054GEAPAPGHHHHHHRPeptide_354GELSYAGHHHHHHRPeptide_654GSYELEGHHHHHHRPeptide_954GALEWWGHHHHHHRPeptide_055GPAPAEGHHHHHHRPeptide_355GSAELYGHHHHHHRPeptide_655GTLDYEGHHHHHHRPeptide_955GELEWEGHHHHHHRPeptide_056GALPALGHHHHHHRPeptide_356GDYTLAGHHHHHHRPeptide_656GSWDYAGHHHHHHRPeptide_956GWYSYSGHHHHHHRPeptide_057GDLPAAGHHHHHHRPeptide_357GTYDALGHHHHHHRPeptide_657GDASWYGHHHHHHRPeptide_957GSYSYWGHHHHHHRPeptide_058GEAPATGHHHHHHRPeptide_358GTADLYGHHHHHHRPeptide_658GSADYWGHHHHHHRPeptide_958GWADWEGHHHHHHRPeptide_059GDADAPGHHHHHHRPeptide_359GLLSYSGHHHHHHRPeptide_659GWYIATGHHHHHHRPeptide_959GEADWWGHHHHHHRPeptide_060GAATLLGHHHHHHRPeptide_360GWLPAPGHHHHHHRPeptide_660GTWTYAGHHHHHHRPeptide_960GDWSLWGHHHHHHRPeptide_061GELSAAGHHHHHHRPeptide_361GPWPALGHHHHHHRPeptide_661GWATYTGHHHHHHRPeptide_961GSWDWLGHHHHHHRPeptide_062GSAELAGHHHHHHRPeptide_362GDYSAEGHHHHHHRPeptide_662GYATWTGHHHHHHRPeptide_962GWWTLPGHHHHHHRPeptide_063GDLTAAGHHHHHHRPeptide_363GSADYEGHHHHHHRPeptide_663GTATYWGHHHHHHRPeptide_963GTLTWWGHHHHHHRPeptide_064GTLDAAGHHHHHHRPeptide_364GEYTATGHHHHHHRPeptide_664GPWELPGHHHHHHRPeptide_964GTYPYYGHHHHHHRPeptide_065GTADALGHHHHHHRPeptide_365GTYDADGHHHHHHRPeptide_665GELPWPGHHHHHHRPeptide_965GWYELPGHHHHHHRPeptide_066GALSLSGHHHHHHRPeptide_366GEATYTGHHHHHHRPeptide_666GPLPWEGHHHHHHRPeptide_966GELPWYGHHHHHHRPeptide_067GTLSATGHHHHHHRPeptide_367GYYPAAGHHHHHHRPeptide_667GYYPAEGHHHHHHRPeptide_967GPLEYWGHHHHHHRPeptide_068GSLTATGHHHHHHRPeptide_368GSYTLTGHHHHHHRPeptide_668GPYEYAGHHHHHHRPeptide_968GDWSWDGHHHHHHRPeptide_069GTASITGHHHHHHRPeptide_369GYAPAYGHHHHHHRPeptide_669GEAPYYGHHHHHHRPeptide_969GYYSLYGHHHHHHRPeptide_070GSATLTGHHHHHHRPeptide_370GTLTYSGHHHHHHRPeptide_670GPAEYYGHHHHHHRPeptide_970GEWPYDGHHHHHHRPeptide_071GAAPAYGHHHHHHRPeptide_371GAAPYYGHHHHHHRPeptide_671GSWSYTGHHHHHHRPeptide_971GEYPWDGHHHHHHRPeptide_072GPLPAPGHHHHHHRPeptide_372GPLELLGHHHHHHRPeptide_672GWLDLPGHHHHHHRPeptide_972GDYEWPGHHHHHHRPeptide_073GYASASGHHHHHHRPeptide_373GPWDAPGHHHHHHRPeptide_673GPWDLLGHHHHHHRPeptide_973GSYDYYGHHHHHHRPeptide_074GSASAYGHHHHHHRPeptide_374GDAPWPGHHHHHHRPeptide_674GPLDWLGHHHHHHRPeptide_974GYYTYTGHHHHHHRPeptide_075GAASYSGHHHHHHRPeptide_375GELDLPGHHHHHHRPeptide_675GDYPYSGHHHHHHRPeptide_975GEWTLYGHHHHHHRPeptide_076GPATLPGHHHHHHRPeptide_376GDLELPGHHHHHHRPeptide_676GSYDYPGHHHHHHRPeptide_976GDWDLYGHHHHHHRPeptide_077GELPAAGHHHHHHRPeptide_377GLLDLLGHHHHHHRPeptide_677GTYTYPGHHHHHHRPeptide_977GEYTWLGHHHHHHRPeptide_078GEAPALGHHHHHHRPeptide_378GWLTAPGHHHHHHRPeptide_678GYYDLAGHHHHHHRPeptide_978GELTYWGHHHHHHRPeptide_079GALPAEGHHHHHHRPeptide_379GTLPWAGHHHHHHRPeptide_679GYADYLGHHHHHHRPeptide_979GDLDYWGHHHHHHRPeptide_080GAAELPGHHHHHHRPeptide_380GTAPWLGHHHHHHRPeptide_680GELPWTGHHHHHHRPeptide_980GWWSAYGHHHHHHRPeptide_081GPLSLAGHHHHHHRPeptide_381GYATAYGHHHHHHRPeptide_681GWLTLLGHHHHHHRPeptide_981GWYSWAGHHHHHHRPeptide_082GEADAPGHHHHHHRPeptide_382GELTLLGHHHHHHRPeptide_682GPWSWAGHHHHHHRPeptide_982GWASYWGHHHHHHRPeptide_083GSADLPGHHHHHHRPeptide_383GEWSAPGHHHHHHRPeptide_683GYAEYTGHHHHHHRPeptide_983GYASWWGHHHHHHRPeptide_084GTLPATGHHHHHHRPeptide_384GSAPWEGHHHHHHRPeptide_684GDADYYGHHHHHHRPeptide_984GSWEYEGHHHHHHRPeptide_085GLLDAAGHHHHHHRPeptide_385GDATWPGHHHHHHRPeptide_685GSAPWWGHHHHHHRPeptide_985GSYEWEGHHHHHHRPeptide_086GALDLAGHHHHHHRPeptide_386GTADWPGHHHHHHRPeptide_686GYYSLTGHHHHHHRPeptide_986GEYTWDGHHHHHHRPeptide_087GEATLAGHHHHHHRPeptide_387GWAELAGHHHHHHRPeptide_687GSYTYLGHHHHHHRPeptide_987GDYEWTGHHHHHHRPeptide_088GLLTASGHHHHHHRPeptide_388GAAELWGHHHHHHRPeptide_688GWAELEGHHHHHHRPeptide_988GTYEWDGHHHHHHRPeptide_089GTASLLGHHHHHHRPeptide_389GPWSLSGHHHHHHRPeptide_689GEAEWLGHHHHHHRPeptide_989GEWPWPGHHHHHHRPeptide_090GSATLLGHHHHHHRPeptide_390GSWSLPGHHHHHHRPeptide_690GALEWEGHHHHHHRPeptide_990GWLPWLGHHHHHHRPeptide_091GAASAWGHHHHHHRPeptide_391GWLSLAGHHHHHHRPeptide_691GEWSLLGHHHHHHRPeptide_991GLLPWWGHHHHHHRPeptide_092GDADADGHHHHHHRPeptide_392GEAELEGHHHHHHRPeptide_692GSWELLGHHHHHHRPeptide_992GWYSYPGHHHHHHRPeptide_093GTADAEGHHHHHHRPeptide_393GYYSASGHHHHHHRPeptide_693GELSLWGHHHHHHRPeptide_993GSYPYWGHHHHHHRPeptide_094GELSASGHHHHHHRPeptide_394GSYSAYGHHHHHHRPeptide_694GWWDAAGHHHHHHRPeptide_994GEYELYGHHHHHHRPeptide_095GSAELSGHHHHHHRPeptide_395GYASYSGHHHHHHRPeptide_695GEYSYSGHHHHHHRPeptide_995GELEYYGHHHHHHRPeptide_096GTLTATGHHHHHHRPeptide_396GELSLEGHHHHHHRPeptide_696GAADWWGHHHHHHRPeptide_996GWWDLPGHHHHHHRPeptide_097GPYSAAGHHHHHHRPeptide_397GPYTLPGHHHHHHRPeptide_697GTYTYTGHHHHHHRPeptide_997GPLDWWGHHHHHHRPeptide_098GSYPAAGHHHHHHRPeptide_398GEWDAAGHHHHHHRPeptide_698GEAEWDGHHHHHHRPeptide_998GWYDAYGHHHHHHRPeptide_099GYASAPGHHHHHHRPeptide_399GAADWEGHHHHHHRPeptide_699GEWSLDGHHHHHHRPeptide_999GWADYYGHHHHHHRPeptide_100GPASYAGHHHHHHRPeptide_400GWLSADGHHHHHHRPeptide_700GDWELSGHHHHHHRPeptide_1000GEYDYEGHHHHHHRPeptide_101GSAPAYGHHHHHHRPeptide_401GSWDLAGHHHHHHRPeptide_701GELTWTGHHHHHHRPeptide_1001GEWTWPGHHHHHHRPeptide_102GAADAYGHHHHHHRPeptide_402GWASLDGHHHHHHRPeptide_702GDLDWTGHHHHHHRPeptide_1002GDWDWPGHHHHHHRPeptide_103GLLPAPGHHHHHHRPeptide_403GDASWLGHHHHHHRPeptide_703GTLDWDGHHHHHHRPeptide_1003GWWTLLGHHHHHHRPeptide_104GPLPALGHHHHHHRPeptide_404GSADLWGHHHHHHRPeptide_704GSYPWPGHHHHHHRPeptide_1004GYLPYYGHHHHHHRPeptide_105GSYTAAGHHHHHHRPeptide_405GTLTWAGHHHHHHRPeptide_705GWYPALGHHHHHHRPeptide_1005GWYSYTGHHHHHHRPeptide_106GYASATGHHHHHHRPeptide_406GTATWLGHHHHHHRPeptide_706GYAPWLGHHHHHHRPeptide_1006GTYSYWGHHHHHHRPeptide_107GSASYSGHHHHHHRPeptide_407GEYPALGHHHHHHRPeptide_707GWWSATGHHHHHHRPeptide_1007GSYTWYGHHHHHHRPeptide_108GALTLPGHHHHHHRPeptide_408GELPAYGHHHHHHRPeptide_708GSWTAWGHHHHHHRPeptide_1008GEWEWAGHHHHHHRPeptide_109GWAPAAGHHHHHHRPeptide_409GPLEYAGHHHHHHRPeptide_709GTASWWGHHHHHHRPeptide_1009GWAEWEGHHHHHHRPeptide_110GELSAPGHHHHHHRPeptide_410GLLPYSGHHHHHHRPeptide_710GSATWWGHHHHHHRPeptide_1010GEAEWWGHHHHHHRPeptide_111GSAELPGHHHHHHRPeptide_411GEWTASGHHHHHHRPeptide_711GEYELPGHHHHHHRPeptide_1011GWWSLEGHHHHHHRPeptide_112GDLTAPGHHHHHHRPeptide_412GTWSAEGHHHHHHRPeptide_712GPLEYEGHHHHHHRPeptide_1012GSWELWGHHHHHHRPeptide_113GTLPADGHHHHHHRPeptide_413GTAEWSGHHHHHHRPeptide_713GWYDAPGHHHHHHRPeptide_1013GELSWWGHHHHHHRPeptide_114GTADLPGHHHHHHRPeptide_414GSATWEGHHHHHHRPeptide_714GDWPAYGHHHHHHRPeptide_1014DGWTLWGHHHHHHRPeptide_115GEADLAGHHHHHHRPeptide_415GDATWTGHHHHHHRPeptide_715GYADWPGHHHHHHRPeptide_1015GTWDLWGHHHHHHRPeptide_116GYAPAPGHHHHHHRPeptide_416GSWTLSGHHHHHHRPeptide_716GPADYWGHHHHHHRPeptide_1016GDLTWWGHHHHHHRPeptide_117GDLSLAGHHHHHHRPeptide_417GEYDAPGHHHHHHRPeptide_717GEYDLLGHHHHHHRPeptide_1017GYYDYPGHHHHHHRPeptide_118GSADLLGHHHHHHRPeptide_418GEAPYDGHHHHHHRPeptide_718GELDLYGHHHHHHRPeptide_1018GEWSWDGHHHHHHRPeptide_119GTLTLAGHHHHHHRPeptide_419GSYDLPGHHHHHHRPeptide_719GDLELYGHHHHHHRPeptide_1019GDWEWSGHHHHHHRPeptide_120GTATLLGHHHHHHRPeptide_420GTLTYPGHHHHHHRPeptide_720GPLPYYGHHHHHHRPeptide_1020GWWPYAGHHHHHHRPeptide_121GWATAAGHHHHHHRPeptide_421GEYTLAGHHHHHHRPeptide_721GSWTYPGHHHHHHRPeptide_1021GWYPWAGHHHHHHRPeptide_122GELSATGHHHHHHRPeptide_422GYLTAEGHHHHHHRPeptide_722GSYPWTGHHHHHHRPeptide_1022GWAPWYGHHHHHHRPeptide_123GEATLSGHHHHHHRPeptide_423GELTAYGHHHHHHRPeptide_723GWYTLAGHHHHHHRPeptide_1023GYAPWWGHHHHHHRPeptide_124GTASLEGHHHHHHRPeptide_424GDLDAYGHHHHHHRPeptide_724GWLTYAGHHHHHHRPeptide_1024GYYTLYGHHHHHHRPeptide_125GDLSADGHHHHHHRPeptide_425GEATLYGHHHHHHRPeptide_725GWATLYGHHHHHHRPeptide_1025GSYEYYGHHHHHHRPeptide_126GDASLDGHHHHHHRPeptide_426GDADYLGHHHHHHRPeptide_726GYATWLGHHHHHHRPeptide_1026GTYDYYGHHHHHHRPeptide_127GTLSLSGHHHHHHRPeptide_427GTAEYLGHHHHHHRPeptide_727GTYELEGHHHHHHRPeptide_1027GDWELYGHHHHHHRPeptide_128GSWSAAGHHHHHHRPeptide_428GTYSLLGHHHHHHRPeptide_728GELDYDGHHHHHHRPeptide_1028GELDYWGHHHHHHRPeptide_129GSASAWGHHHHHHRPeptide_429GSYTLLGHHHHHHRPeptide_729GDLDYEGHHHHHHRPeptide_1029GYYPWPGHHHHHHRPeptide_130GTAPAYGHHHHHHRPeptide_430GLLSYTGHHHHHHRPeptide_730GPYDYPGHHHHHHRPeptide_1030GWWTYAGHHHHHHRPeptide_131GPYSASGHHHHHHRPeptide_431GWYSAAGHHHHHHRPeptide_731GEWSAYGHHHHHHRPeptide_1031GWYTWAGHHHHHHRPeptide_132GSYPASGHHHHHHRPeptide_432GWASYAGHHHHHHRPeptide_732GEYSWAGHHHHHHRPeptide_1032GWATYWGHHHHHHRPeptide_133GPASYSGHHHHHHRPeptide_433GYASAWGHHHHHHRPeptide_733GSYEWAGHHHHHHRPeptide_1033GEWDYDGHHHHHHRPeptide_134GSAPYSGHHHHHHRPeptide_434GAASYWGHHHHHHRPeptide_734GEASWYGHHHHHHRPeptide_1034GDWEYDGHHHHHHRPeptide_135GALSAYGHHHHHHRPeptide_435GSAEYEGHHHHHHRPeptide_735GSAEWYGHHHHHHRPeptide_1035GEYEWPGHHHHHHRPeptide_136GSYDAAGHHHHHHRPeptide_436GDADYDGHHHHHHRPeptide_736GDATYWGHHHHHHRPeptide_1036GDYDWEGHHHHHHRPeptide_137GSADAYGHHHHHHRPeptide_437GTADYEGHHHHHHRPeptide_737GWYSLSGHHHHHHRPeptide_1037GTYEWEGHHHHHHRPeptide_138GTYTAAGHHHHHHRPeptide_438GELSYSGHHHHHHRPeptide_738GWLSYSGHHHHHHRPeptide_1038GWWSYSGHHHHHHRPeptide_139GYATATGHHHHHHRPeptide_439GSYDLTGHHHHHHRPeptide_739GSWSLYGHHHHHHRPeptide_1039GSYSWWGHHHHHHRPeptide_140GTATAYGHHHHHHRPeptide_440GTYTLTGHHHHHHRPeptide_740GWWPAPGHHHHHHRPeptide_1040GWWELPGHHHHHHRPeptide_141GAATYTGHHHHHHRPeptide_441GTLTYTGHHHHHHRPeptide_741GPWPAWGHHHHHHRPeptide_1041GEWPWLGHHHHHHRPeptide_142GELPAPGHHHHHHRPeptide_442GEWPAPGHHHHHHRPeptide_742GWAPWPGHHHHHHRPeptide_1042GWLEWPGHHHHHHRPeptide_143GLLPALGHHHHHHRPeptide_443GEAPWPGHHHHHHRPeptide_743GYYTLPGHHHHHHRPeptide_1043GPWEEWGHHHHHHRPeptide_144GSYTASGHHHHHHRPeptide_444GPWSLPGHHHHHHRPeptide_744GSWTYTGHHHHHHRPeptide_1044GELPWWGHHHHHHRPeptide_145GSATYSGHHHHHHRPeptide_445GYYSAPGHHHHHHRPeptide_745GSYTWTGHHHHHHRPeptide_1045GYYEWAGHHHHHHRPeptide_146GPADLLGHHHHHHRPeptide_446GSYPAYGHHHHHHRPeptide_746GWLELPGHHHHHHRPeptide_1046GWAEYYGHHHHHHRPeptide_147GDADLPGHHHHHHRPeptide_447GYAPYSGHHHHHHRPeptide_747GELPWLGHHHHHHRPeptide_1047GYAEYWGHHHHHHRPeptide_148GELTAPGHHHHHHRPeptide_448GSAPYYGHHHHHHRPeptide_748GEYSYPGHHHHHHRPeptide_1048GWYSYLGHHHHHHRPeptide_149GEATLPGHHHHHHRPeptide_449GELELPGHHHHHHRPeptide_749GEWDLPGHHHHHHRPeptide_1049GEYEYIGHHHHHHRPeptide_150GTLSLPGHHHHHHRPeptide_450GLLELLGHHHHHHRPeptide_750GELPWDGHHHHHHRPeptide_1050GEWDWPGHHHHHHRPeptide_151GPWSAAGHHHHHHRPeptide_451GDWPALGHHHHHHRPeptide_751GLLDLWGHHHHHHRPeptide_1051GWWDLLGHHHHHHRPeptide_152GSWPAAGHHHHHHRPeptide_452GDAPWLGHHHHHHRPeptide_752GEYDAYGHHHHHHRPeptide_1052GSWDYYGHHHHHHRPeptide_153GWASAPGHHHHHHRPeptide_453GYYDAAGHHHHHHRPeptide_753GEADYYGHHHHHHRPeptide_1053GSYDYWGHHHHHHRPeptide_154GPASAWGHHHHHHRPeptide_454GAADYYGHHHHHHRPeptide_754GDAEYYGHHHHHHRPeptide_1054GWYTYTGHHHHHHRPeptide_155GAAELEGHHHHHHRPeptide_455GELDLLGHHHHHHRPeptide_755GWWTAPGHHHHHHRPeptide_1055GYYTWTGHHHHHHRPeptide_156GELSLAGHHHHHHRPeptide_456GEWTAPGHHHHHHRPeptide_756GTAPWWGHHHHHHRPeptide_1056GTYTYWGHHHHHHRPeptide_157GSAELLGHHHHHHRPeptide_457GDWPADGHHHHHHRPeptide_757GYYTLTGHHHHHHRPeptide_1057GEWTLWGHHHHHHRPeptide_158GTLDLAGHHHHHHRPeptide_458GEAPWTGHHHHHHRPeptide_758GTYTLYGHHHHHHRPeptide_1058GWLEWTGHHHHHHRPeptide_159GDATLLGHHHHHHRPeptide_459GTAPWEGHHHHHHRPeptide_759GEWTLLGHHHHHHRPeptide_1059GELTWWGHHHHHHRPeptide_160GSLSLLGHHHHHHRPeptide_460GALTWLGHHHHHHRPeptide_760GDWDLLGHHHHHHRPeptide_1060GTLEWWGHHHHHHRPeptide_161GWADAAGHHHHHHRPeptide_461GYYSATGHHHHHHRPeptide_761GTWELLGHHHHHHRPeptide_1061GEYPYYGHHHHHHRPeptide_162GELDASGHHHHHARPeptide_462GTYSAYGHHHHHHRPeptide_762GELTLWGHHHHHHRPeptide_1062GWWSAWGHHHHHHRPeptide_163GDLTADGHHHHHHRPeptide_463GSYTAYGHHHHHHRPeptide_763GLLEWTGHHHHHHRPeptide_1063GEWSWEGHHHHHHRPeptide_164GDADLTGHHHHHHRPeptide_464GYATYSGHHHHHHRPeptide_764GTLEWLGHHHHHHRPeptide_1064GSWEWEGHHHHHHRPeptide_165GEATLTGHHHHHHRPeptide_465GTLELEGHHHHHHRPeptide_765GWAEWAGHHHHHHRPeptide_1065GWWSYPGHHHHHHRPeptide_166GSYPAPGHHHHHHRPeptide_466GPYDLPGHHHHHHRPeptide_766GAAEWWGHHHHHHRPeptide_1066GYYDYDGHHHHHHRPeptide_167GYAPALGHHHHHHRPeptide_467GEAEWAGHHHHHHRPeptide_767GDYSYDGHHHHHHRPeptide_1067GPWSYWGHHHHHHRPeptide_168GTLSLTGHHHHHHRPeptide_468GAAEWEGHHHHHHRPeptide_768GSYDYDGHHHHHHRPeptide_1068GSWPYWGHHHHHHRPeptide_169GWASATGHHHHHHRPeptide_469GEWSLAGHHHHHHRPeptide_769GEYSYTGHHHHHHRPeptide_1069GPYSWWGHHHHHHRPeptide_170GSATAWGHHHHHHRPeptide_470GSWELAGHHHHHHRPeptide_770GWWSLAGHHHHHHRPeptide_1070GSYPWWGHHHHHHRPeptide_171GDAPAYGHHHHHHRPeptide_471GWASLEGHHHHHHRPeptide_771GEAEWEGHHHHHHRPeptide_1071GEWEYLGHHHHHHRPeptide_172GPADAYGHHHHHHRPeptide_472GELSWAGHHHHHHRPeptide_772GELSWEGHHHHHHRPeptide_1072GEYEWLGHHHHHHRPeptide_173GSWSASGHHHHHHRPeptide_473GSAELWGHHHHHHRPeptide_773GEWTLDGHHHHHHRPeptide_1073GELEYWGHHHHHHRPeptide_174GSYTAPGHHHHHHRPeptide_474GDWTLAGHHHHHHRPeptide_774GDASWWGHHHHHHRPeptide_1074GWWDAYGHHHHHHRPeptide_175GTAPYSGHHHHHHRPeptide_475GTADWLGHHHHHHRPeptide_775GSADWWGHHHHHHRPeptide_1075GWADYWGHHHHHHRPeptide_176GSATYPGHHHHHHRPeptide_476GSYSYSGHHHHHHRPeptide_776GWWTATGHHHHHHRPeptide_1076GYADWWGHHHHHHRPeptide_177GYATLAGHHHHHHRPeptide_477GPYTLLGHHHHHHRPeptide_777GWATWTGHHHHHHRPeptide_1077GEWDYEGHHHHHHRPeptide_178GAATYLGHHHHHHRPeptide_478GLLTYPGHHHHHHRPeptide_778GTATWWGHHHHHHRPeptide_1078GDWEYEGHHHHHHRPeptide_179GEYSAAGHHHHHHRPeptide_479GDWSAEGHHHHHHRPeptide_779GEWPAYGHHHHHHRPeptide_1079GEYEWDGHHHHHHRPentide_180GSAEYAGHHHHHHRPeptide_480GSADWEGHHHHHHRPeptide_780GEYPWAGHHHHHHRPeptide_1080GYYPWLGHHHHHHRPeptide_181GDATAYGHHHHHHRPeptide_481GEWTATGHHHHHHRPeptide_781GYAPWEGHHHHHHRPeptide_1081GWWSYTGHHHHHHRPeptide_182GSYSLAGHHHHHHRPeptide_482GTWDADGHHHHHHRPeptide_782GEAPYWGHHHHHHRPeptide_1082GSYTWWGHHHHHHRPeptide_183GYASLSGHHHHHHRPeptide_483GDADWTGHHHHHHRPeptide_783GWYSLPGHHHHHHRPeptide_1083GDWPYYGHHHHHHRPeptide_184GSLSAYGHHHHHHRPeptide_484GTADWDGHHHHHHRPeptide_784GWLSYPGHHHHHHRPeptide_1084GPWDYYGHHHHHHRPeptide_185GPWPAAGHHHHHHRPeptide_485GSLSWDGHHHHHHRPeptide_785GPWSLYGHHHHHHRPeptide_1085GWWPAWGHHHHHHRPeptide_186GWAPAPGHHHHHHRPeptide_486GSWTLTGHHHHHHRPeptide_786GSYPWLGHHHHHHRPeptide_1086GWYTLYGHHHHHHRPeptide_187GAAPWPGHHHHHHRPeptide_487GWYPAYGHHHHHHRPeptide_787GPLSWYGHHHHHHRPeptide_1087GWLELWGHHHHHHRPeptide_188GDASYSGHHHHHHRPeptide_488GAAPWYGHHHHHHRPeptide_788GYYSAYGHHHHHHRPeptide_1088GEYSWYGHHHHHHRPeptide_189GTYTASGHHHHHHRPeptide_489GEYSLPGHHHHHHRPeptide_789GYLELEGHHHHHHRPeptide_1089GSYEYWGHHHHHHRPeptide_190GSYTATGHHHHHHRPeptide_490GSYELPGHHHHHHRPeptide_790GELELYGHHHHHHRPeptide_1090GYYTWDGHHHHHHRPeptide_191GTASYTGHHHHHHRPeptide_491GELPYSGHHHHHHRPeptide_791GSYPWDGHHHHHHRPeptide_1091GDYTYWGHHHHHHRPeptide_192GSATYTGHHHHHHRPeptide_492GDYTLPGHHHHHHRPeptide_792GTWTYPGHHHHHHRPeptide_1092GTYDWYGHHHHHHRPeptide_193GELPALGHHHHHHRPeptide_493GTLPYDGHHHHHHRPeptide_793GTYTWPGHHHHHHRPeptide_1093GEWDLWGHHHHHHRPeptide_194GLLPAEGHHHHHHRPeptide_494GYAELLGHHHHHHRPeptide_794GWYDALGHHHHHHRPeptide_1094GELDWWGHHHHHHRPeptide_195GPAELLGHHHHHHRPeptide_495GALELYGHHHHHHRPeptide_795GWADLYGHHHHHHRPeptide_1095GWYPWPGHHHHHHRPeptide_196GPLDAEGHHHHHHRPeptide_496GEYDALGHHHHHHRPeptide_796GYADWLGHHHHHHRPeptide_1096GPWPYWGHHHHHHRPeptide_197GEADLPGHHHHHHRPeptide_497GELDYAGHHHHHHRPeptide_797GELDYEGHHHHHHRPeptide_1097GPYPWWGHHHHHHRPeptide_198GDAELPGHHHHHHRPeptide_498GEADLYGHHHHHHRPeptide_798GDLEYEGHHHHHHRPeptide_1098GWATWWGHHHHHHRPeptide_199GTLTLPGHHHHHHRPeptide_499GYYPAPGHHHHHHRPeptide_799GWADYDGHHHHHHRPeptide_1099GEWTWEGHHHHHHRPeptide_200GWATAPGHHHHHHRPeptide_500GSYDLLGHHHHHHRPeptide_800GDADYWGHHHHHHRPeptide_1100GDWEWDGHHHHHHRPeptide_201GAAPWTGHHHHHHRPeptide_501GDLSLYGHHHHHHRPeptide_801GEWTYAGHHHHHHRPeptide_1101GTWEWEGHHHHHHRPeptide_202GELTLAGHHHHHHRPeptide_502GTLTLYGHHHHHHRPeptide_802GEYTAWGHHHHHHRPeptide_1102GYYELYGHHHHHHRPeptide_203GDLDLAGHHHHHHRPeptide_503GWATYAGHHHHHHRPeptide_803GYAEWTGHHHHHHRPeptide_1103GYLEYYGHHHHHHRPeptide_204GEATLLGHHHHHHRPeptide_504GYATAWGHHHHHHRPeptide_804GTAEWYGHHHHHHRPeptide_1104GSWSWWGHHHHHHRPeptide_205GTLSLLGHHHHHHRPeptide_505GAATYWGHHHHHHRPeptide_805GWYSLTGHHHHHHRPeptide_1105GEYDYYGHHHHHHRPeptide_206GAAEWAGHHHHHHRPeptide_506GEADYDGHHHHHHRPeptide_806GSWTYLGHHHHHHRPeptide_1106GWYPWTGHHHHHHRPeptide_207GPWSASGHHHHHHRPeptide_507GDADYEGHHHHHHRPeptide_807GTYSLWGHHHHHHRPeptide_1107GWWEYAGHHHHHHRPeptide_208GWLSAAGHHHHHHRPeptide_508GTAEYEGHHHHHHRPeptide_808GSYTWLGHHHHHHRPeptide_1108GWAEYWGHHHHHHRPeptide_209GWASLAGHHHHHHRPeptide_509GEYSLTGHHHHHHRPeptide_809GSLTWYGHHHHHHRPeptide_1109GYAEWWGHHHHHHRPeptide_210GALSAWGHHHHHHRPeptide_510GTYSLEGHHHHHHRPeptide_810GPLDYYGHHHHHHRPeptide_1110GWYSWLGHHHHHHRPeptide_211GSAELEGHHHHHHRPeptide_511GELSYTGHHHHHHRPeptide_811GEWSYSGHHHHHHRPeptide_1111GWLSWYGHHHHHHRPeptide_212GDLDADGHHHHHHRPeptide_512GWYSASGHHHHHHRPeptide_812GSYEWSGHHHHHHRPeptide_1112GEWEYEGHHHHHHRPeptide_213GTYPAPGHHHHHHRPeptide_513GSWSYAGHHHHHHRPeptide_813GDWTYSGHHHHHHRPeptide_1113GWWSYDGHHHHHHRPeptide_214GELSLSGHHHHHHRPeptide_514GWASYSGHHHHHHRPeptide_814GTYDWSGHHHHHHRPeptide_1114GWYSWDGHHHHHHRPeptide_215GSLELSGHHHHHHRPeptide_515GSYSWAGHHHHHHRPeptide_815GDYPYDGHHHHHHRPeptide_1115GSYDWWGHHHHHHRPeptide_216GTLTLTGHHHHHHRPeptide_516GSASYWGHHHHHHRPeptide_816GSWPWPGHHHHHHRPeptide_1116GTYTWWGHHHHHHRPeptide_217GSWDAAGHHHHHHRPeptide_517GTLPWPGHHAHHHRPeptide_817GWWPALGHHHHHHRPeptide_1117GEWPYYGHHHHHHRPeptide_218GWASADGHHHHHHRPeptide_518GYYTAPGHHHHHHRPeptide_818GWAPWLGHHHHHHRPeptide_1118GEYPWYGHHHHHHRPeptide_219GTWTAAGHHHHHHRPeptide_519GYAPYTGHHHHHHRPeptide_819GYY7LIGHHHHHHRPeptide_1119GWLDYYGHHHHHHRPeptide_220GTATAWGHHHHHHRPeptide_520GTAPYYGHHHHHHRPeptide_820GEWELPGHHHHHHRPeptide_1120GWYTYEGHHHHHHRPeptide_221GAATWTGHHHHHHRPeptide_521GEWPALGHHHHHHRPeptide_821GPWELEGHHHHHHRPeptide_1121GYYDWDGHHHHHHRPeptide_222GEYPAAGHHHHHHRPeptide_522GELPAWGHHHHHHRPeptide_822GELPWEGHHHHHHRPeptide_1122GEYTYWGHHHHHHRPeptide_223GEAPAYGHHHHHHRPeptide_523GEAPWLGHHHHHHRPeptide_823GEYEYAGHHHHHHRPeptide_1123GTWEYYGHHHHHHRPeptide_224GPAEYAGHHHHHHRPeptide_524GPLSLWGHHHHHHRPeptide_824GWWDAPGHHHHHHRPeptide_1124GPWSWWGHHHHHHRPeptide_225GYLSAPGHHHHHHRPeptide_525GYAEYAGHHHHHHRPeptide_825GWAPWDGHHHHHHRPeptide_1125GDYDYWGHHHHHHRPeptide_226GSYPALGHHHHHHRPeptide_526GAAEYYGHHHHHHRPeptide_826GDAPWWGHHHHHHRPeptide_1126GEWELWGHHHHHHRPeptide_227GYASLPGHHHHHHRPeptide_527GSYPYSGHHHHHHRPeptide_827GYYSIEGHHHHHHRPeptide_1127GWLEWEGHHHHHHRPeptide_228GPLSAYGHHHHHHRPeptide_528GYYSLAGHHHHHHRPeptide_828GSYEYLGHHHHHHRPeptide_1128GELEWWGHHHHHHRPeptide_229GALSYPGHHHHHHRPeptide_529GEWDAPGHHHHHHRPeptide_829GSLEYYGHHHHHHRPeptide_1129GEWDWEGHHHHHHRPeptide_230GSWTASGHHHHHHRPeptide_530GEAPWDGHHHHHHRPeptide_830GTYDYLGHHHHHHRPeptide_1130GWYPWLGHHHHHHRPeptide_231GSYPADGHHHHHHRPeptide_531GDAEWPGHHHHHHRPeptide_831GEWDLLGHHHHHHRPeptide_1131GWLPYWGHHHHHHRPeptide_232GPASYDGHHHHHHRPeptide_532GPAEWDGHHHHHHRPeptide_832GDWELLGHHHHHHRPeptide_1132GYLPWWGHHHHHHRPeptide_233GSADYPGHHHHHHRPeptide_533GDWSLPGHHHHHHRPeptide_833GELDLWGHHHHHHRPeptide_1133GSWTWWGHHHHHHRPeptide_234GTYTAPGHHHHHHRPeptide_534GSWDLPGHHHHHHRPeptide_834GPLPYWGHHHHHHRPeptide_1134GYYEYEGHHHHHHRPeptide_235GDADAYGHHHHHHRPeptide_535GTLTWPGHHHHHHRPeptide_835GEYSYDGHHHHHHRPeptide_1135GWWDYPGHHHHHHRPeptide_236GAADYDGHHHHHHRPeptide_536GWADLLGHHHHHHRPeptide_836GSYEYDGHHHHHHRPeptide_1136GWYDWPGHHHHHHRPeptide_237GSYTLAGHHHHHHRPeptide_537GSYDAYGHHHHHHRPeptide_837GTYDYDGHHHHHHRPeptide_1137GPYDWWGHHHHHHRPeptide_238GYASLTGHHHHHHRPeptide_538GSADYYGHHHHHHRPeptide_838GWWTLAGHHHHHHRPeptide_1138GYYPYYGHHHHHHRPeptide_239GALTYSGHHHHHHRPeptide_539GYYTATGHHHHHHRPeptide_839GWATWLGHHHHHHRPeptide_1139GWYSWEGHHHHHHRPeptide_240GSATYLGHHHHHHRPeptide_540GTYTAYGHHHHHHRPeptide_840GYYPAYGHHHHHHRPeptide_1140GEWSWYGHHHHHHRPeptide_241GEYSASGHHHHHHRPeptide_541GYATYTGHHHHHHRPeptide_841GYAPYYGHHHHHHRPeptide_1141GEYSWWGHHHHHHRPeptide_242GEASYSGHHHHHHRPeptide_542GPYELPGHHHHHHRPeptide_842GELDWDGHHHHHHRPeptide_1142GSWEYWGHHHHHHRPeptide_243GTYSADGHHHHHHRPeptide_543GEWTALGHHHHHHRPeptide_843GDLDWEGHHHHHHRPeptide_1143GSYEWWGHHHHHHRPeptide_244GSYDATGHHHHHHRPeptide_544GDWDALGHHHHHHRPeptide_844GTLEWEGHHHHHHRPeptide_1144GTWDYWGHHHHHHRPeptide_245GTATYTGHHHHHHRPeptide_545GTWELAGHHHHHHRPeptide_845GWWSAEGHHHHHHRPeptide_1145GWWPWPGHHHHHHRPeptide_246GPLTLLGHHHHHHRPeptide_546GELTWAGHHHHHHRPeptide_846GEWSAWGHHHHHHRPeptide_1146GPWPWWGHHHHHHRPeptide_247GPWPASGHHHHHHRPeptide_547GTLEWAGHHHHHHRPeptide_847SGWEWAGHHHHHHRPeptide_1147GYYEWLGHHHHHHRPeptide_248GSAPWPGHHHHHHRPeptide_548GEATWLGHHHHHHRPeptide_848GWASWEGHHHHHHRPeptide_1148GWLEYYGHHHHHHRPeptide_249GWLPAAGHHHHHHRPeptide_549GDADWLGHHHHHHRPeptide_849GEASWWGHHHHHHRPeptide_1149GWYDYEGHHHHHHRPeptide_250GAAPWLGHHHHHHRPeptide_550GTAEWLGHHHHHHRPeptide_850GSAEWWGHHHHHHRPeptide_1150GEWDYYGHHHHHHRPeptide_251GELPAEGHHHHHHRPeptide_551GSWTLLGHHHHHHRPeptide_851GDATWWGHHHHHHRPeptide_1151GWWEWAGHHHHHHRPeptide_252GEAELPGHHHHHHRPeptide_552GWWSAAGHHHHHHRPeptide_852GTADWWGHHHHHHRPeptide_1152GEWEWEGHHHHHHRPeptide_253GTLDLPGHHHHHHRPeptide_553GWASAWGHHHHHHRPeptide_853GWYTLPGHHHHHHRPeptide_1153GDWSWWGHHHHHHRPeptide_254GLLSLLGHHHHHHRPeptide_554GAASWWGHHHHHHRPeptide_854GTYPWLGHHHHHHRPeptide_1154GSWDWWGHHHHHHRPeptide_255GWADAPGHHHHHHRPeptide_555GSYSYTGHHHHHHRPeptide_855GYYTAYGHHHHHHRPeptide_1155GWWTWTGHHHHHHRPeptide_256GAADWPGHHHHHHRPeptide_556GPLDLYGHHHHHHRPeptide_856GEWPYSGHHHHHHRPeptide_1156GWYEWPGHHHHHHRPeptide_257GELDLAGHHHHHHRPeptide_557GEAEWSGHHHHHHRPeptide_857GSYEWPGHHHHHHRPeptide_1157GEWPWYGHHHHHHRPeptide_258GDAELLGHHHHHHRPeptide_558GSAEWEGHHHHHHRPeptide_858GDWTYPGHHHHHHRPeptide_1158GEYPWWGHHHHHHRPeptide_259GYLPAPGHHHHHHRPeptide_559GDADWDGHHHHHHRPeptide_859GWAELYGHHHHHHRPeptide_1159GPYEWWGHHHHHHRPeptide_260GPYPALGHHHHHHRPeptide_560GEWSLSGHHHHHHRPeptide_860GYAEWLGHHHHHHRPeptide_1160GYYSYWGHHHHHHRPeptide_261GSLDLLGHHHHHHRPeptide_561GDWTLSGHHHHHHRPeptide_861GELEYEGHHHHHHRPeptide_1161GWWDLYGHHHHHHRPeptide_262GSAPWTGHHHHHHRPeptide_562GTLSWDGHHHHHHRPeptide_862GYYSYSGHHHHHHRPeptide_1162GWLDYWGHHHHHHRPeptide_263GWATALGHHHHHHRPeptide_563GTWTLTGHHHHHHRPeptide_863GPWPWPGHHHHHHRPeptide_1163GWWTYEGHHHHHHRPeptide_264GALTAWGHHHHHHRPeptide_564GDLDYPGHHHHHHRPeptide_864GEWDYAGHHHHHHRPeptide_1164GWYEWTGHHHHHHRPeptide_265GAATWLGHHHHHHRPeptide_565GWYSAPGHHHHHHRPeptide_865GWADYEGHHHHHHRPeptide_1165GEWTWYGHHHHHHRPeptide_266GELTAEGHHHHHHRPeptide_566GPWSYAGHHHHHHRPeptide_866GEADWYGHHHHHHRPeptide_1166GEYTWWGHHHHHHRPeptide_267GTAELEGHHHHHHRPeptide_567GSAPYWGHHHHHHRPeptide_867GDAEWYGHHHHHHRPeptide_1167GWWPWLGHHHHHHRPeptide_268GTLSLEGHHHHHHRPeptide_568GELEYAGHHHHHHRPeptide_868GDWSYLGHHHHHHRPeptide_1168GWLPWWGHHHHHHRPeptide_269GDLSLDGHHHHHHRPeptide_569GYAELEGHHHHHHRPeptide_869GDYSLWGHHHHHHRPeptide_1169GWYEYEGHHHHHHRPeptide_270GEWSAAGHHHHHHRPeptide_570GBAEYLGHHHHHHRPeptide_870GSWDYLGHHHHHHRPeptide_1170GYYEWEGHHHHHHRPeptide_271GEASAWGHHHHHHRPeptide_571GEYSLLGHHHHHHRPeptide_871GSYDWLGHHHHHHRPeptide_1171GEWEYYGHHHHHHRPeptide_272GSABWAGHHHHHHRPeptide_572GSYELLGHHHHHHRPeptide_872GDESWYGHHHHHHRPeptide_1172GEYEYWGHHHHHHRPeptide_273GAASWEGHHHHHHRPeptide_573GTYDLLGHHHHHHRPeptide_873GWYTLTGHHHHHHRPeptide_1173GPWDWWGHHHHHHRPeptide_274GWLSASGHHHHHHRPeptide_574GWYDAAGHHHHHHRPeptide_874GTLTYWGHHHHHHRPeptide_1174GWWTLWGHHHHHHRPeptide_275GSWSALGHHHHHHRPeptide_575GWADYAGHHHHHHRPeptide_875GYYELPGHHHHHHRPeptide_1175GWLTWWGHHHHHHRPeptide_276GTLPAYGHHHHHHRPeptide_576GAADWYGHHHHHHRPeptide_876GELPYYGHHHHHHRPeptide_1176GWYPYYGHHHHHHRPeptide_277GSASWDGHHHHHHRPeptide_577GLLPWPGHHHHHHRPeptide_877GPLEYYGHHHHHHRPeptide_1177GYYPYWGHHHHHHRPeptide_278GTWTASGHHHHHHRPeptide_578GPLPWLGHHHHHHRPeptide_878GEWSYTGHHHHHHRPeptide_1178GWWSWEGHHHHHHRPeptide_279GSWTATGHHHHHHRPeptide_579GDAEYEGHHHHHHRPeptide_879GDWDYSGHHHHHHRPeptide_1179GEWSWWGHHHHHHRPeptide_280GEYSAPGHHHHHHRPeptide_580GELDYSGHHHHHHRPeptide_880GDYDWSGHHHHHHRPeptide_1180GYYTYWGHHHHHHRPeptide_281GDYTAPGHHHHHHRPeptide_581GSLDYEGHHHHHHRPeptide_881GSYDWDGHHHHHHRPeptide_1181GWYEWLGHHHHHHRPeptide_282GTYPADGHHHHHHRPeptide_582GTLTYEGHHHHHHRPeptide_882GEYPYDGHHHHHHRPeptide_1182GYLEWWGHHHHHHRPeptide_283GTADYPGHHHHHHRPeptide_583GDYDLTGHHHHHHRPeptide_883GYYDLLGHHHHHHRPeptide_1183GWWDYEGHHHHHHRPeptide_284GAAEYLGHHHHHHRPeptide_584GPYSYPGHHHHHHRPeptide_884GPWEWAGHHHHHHRPeptide_1184GDWEYWGHHHHHHRPeptide_285GSYSLPGHHHHHHRPeptide_585GYYPALGHHHHHHRPeptide_885GEAPWWGHHHHHHRPeptide_1185GEYDWWGHHHHHHRPeptide_286GPLSYSGHHHHHHRPeptide_586GYLPAYGHHHHHHRPeptide_886GWWSLPGHHHHHHRPeptide_1186GEWPWWGHHHHHHRPeptide_287GSLSYPGHHHHHHRPeptide_587GALPYYGHHHHHHRPeptide_887GEYTLYGHHHHHHRPeptide_1187GPWEWWGHHHHHHRPeptide_288GLLSYAGHHHHHHRPeptide_588GWYSATGHHHHHHRPeptide_888GYLEYTGHHHHHHRPeptide_1188GYYSWWGHHHHHHRPeptide_289GEYDAAGHHHHHHRPeptide_589GSWTYAGHHHHHHRPeptide_889GSWPWLGHHHHHHRPeptide_1189GWWTWEGHHHHHHRPeptide_290GYADAEGHHHHHHRPeptide_590GTYSAWGHHHHHHRPeptide_890GPLSWWGHHHHHHRPeptide_1190GEWTWWGHHHHHHRPeptide_291GAADYEGHHHHHHRPeptide_591GWASYTGHHHHHHRPeptide_891GWYSAYGHHHHHHRPeptide_1191GDWDWWGHHHHHHRPeptide_292GSYDLAGHHHHHHRPeptide_592GSYTAWGHHHHHHRPeptide_892GEWELLGHHHHHHRPeptide_1192GYYDYWGHHHHHHRPeptide_293GDASYLGHHHHHHRPeptide_593GTASYWGHHHHHHRPeptide_893GELELWGHHHHHHRPeptide_1193GWWEYEGHHHHHHRPeptide_294GSADLYGHHHHHHRPeptide_594GSATYWGHHHHHHRPeptide_894GLLEWEGHHHHHHRPeptide_1194GWYEWEGHHHHHHRPeptide_295GTYTLAGHHHHHHRPeptide_595GPLPWDGHHHHHHRPeptide_895GSYEYEGHHHHHHRPeptide_1195GEWEYWGHHHHHHRPeptide_296GTLTAYGHHHHHHRPeptide_596GYYDAPGHHHHHHRPeptide_896GSWDWPGHHHHHHRPeptide_1196GEYEWWGHHHHHHRPeptide_297GALTYTGHHHHHHRPeptide_597GSWSYSGHHHHHHRPeptide_897GTWTWPGHHHHHHRPeptide_1197GEWDWWGHHHHHHRPeptide_298GTATYLGHHHHHHRPeptide_598GWLTLPGHHHHHHRPeptide_898GWWDLAGHHHHHHRPeptide_1198GDWEWWGHHHHHHRPeptide_299GTYSAEGHHHHHHRPeptide_599GPWTLLGHHHHHHRPeptide_899GDWELEGHHHHHHRPeptide_1199GWYEYYGHHHHHHRPeptide_300GSADYDGHHHHHHRPeptide_600GLLPWTGHHHHHHRPeptide_900GELDWEGHHHHHHRPeptide_1200GYYEYWGHHHHHHR

10. Screening of Signal Peptides Capable of Increasing Aflibercept Production Using Aflibercept Target Library

Aflibercept as a model target protein was cloned into the signal peptide/SP-tag library to construct a target protein library. Three days after expression of the library in HEK 293 cells, the cell culture media were collected and the expressed protein was isolated and purified using His tags. After treatment of the protein samples with trypsin, the peak area values were calculated by LC-MS/MS to determine the relative amounts of the SP-tags. To effectively analyze 1,200 barcoding peptides designed for signal peptide screening and 1,200 synthesized internal standards (a total of 2,400 peptides), the peptides were divided into a total of 16 groups, 150 target peptides per group, and MRM analysis was performed. Each group (150 target peptides) consisted of 75 barcoding peptides and 75 standard peptides. The global standard peptide was analyzed together for normalization to minimize differences between the 16 groups (FIG. 16). The system was found to be stable for the 16 groups, with a coefficient of variation (CV) of 17.26% (FIG. 16). The 16 groups (2,400 barcoding and standard peptides) were analyzed by LC-MS/MS. The peak area values were corrected with the global standard and are graphically shown inFIG. 17. The high values were selected as primary hits. To validate the screening system and select secondary hits, plasmids corresponding to the top 24 peptides and the bottom 24 peptides were individually expressed and their expression levels were compared by Western blot (FIG. 18). For quantitative analysis, the individually expressed samples were quantified using an OCTET system and compared in duplicate (FIG. 18). Based on these results, 14 signal peptides matched to the 14 SP-tags with high expression levels were selected (FIG. 19).

Although the particulars of the present invention have been described in detail, it will be obvious to those skilled in the art that such particulars are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.