Water-soluble fusion polypeptide comprising internalin protein-derived chimeric peptide fused to n-terminus thereof and use thereof

Abstract

The present invention relates to: a water-soluble fusion polypeptide comprising an internalin protein-derived chimeric peptide fused to the N-terminus thereof; and use thereof and, more specifically, to a fusion polypeptide and use thereof, the fusion polypeptide having one structure selected from the group consisting of: a first structure in which an internalin protein-derived chimeric peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a leucine-rich repeat (LRR) scaffold consisting of the amino acid sequence of SEQ ID NO: 2 are fused; a second structure in which a lysine (Lys)-glutamate (Glu) ionic bond pair is additionally introduced into the LRR peptide of the first structure; a third structure in which an arginine (Arg)-Glu ionic bond pair is additionally introduced into the LRR peptide of the first structure; a fourth structure in which the C-terminus of the LRR peptide of the first structure is substituted with a sequence derived from Shigella IpaH7.8; and a fifth structure in which amino acids at positions 176 to 179 at the C-terminus of the LRR peptide of the first structure are substituted with Xn (X is an arbitrary amino acid, and n is an integer from 12 to 18).

Description

Water-soluble fusion polypeptide in which a chimeric peptide derived from internalin protein is fused to the amino terminus and uses thereof

[0001] The present invention relates to a water-soluble fusion polypeptide comprising a chimeric peptide derived from the amino terminus of an internalin protein at the amino terminus, and to the use thereof.

[0002]

[0003] This project was carried out with support from the National Research Foundation of Korea’s Biomedical Technology Development Project (Project No.: 00235511) and the Korea Institute of Technology Information Promotion (KITIPA)’s Startup Growth Technology Development (Project No.: 00553842).

[0004] The most primitive life forms in the classification of vertebrates are jawless fish, known as jawless agnathas, of which only two species remain today: lampreys and hagfish. In 2004, it was first discovered that the adaptive immune system of these two organisms utilizes an antigen receptor called the variable lymphocyte receptor (VLR) rather than the immunoglobulin (Ig) type found in conventional vertebrates. Furthermore, DNA sequence analysis and studies of protein tertiary structures revealed that the VLR possesses a leucine-rich repeat (LRR) fold. Although LRR-type proteins are found in almost all species ranging from prokaryotes to eukaryotes, the VLRs of lampreys and hagfish possess diversity similar to the complementary determining region (CDR) of conventional Ig antibodies, as amino acid residues in the β-strand and loop regions of the LRR domain are located at random positions (~10 14It is a natural adaptive immune system of non-Ig folding type, which is theoretically capable of recognizing almost all antigens and is almost the only known to date. This characteristic of VLR is highly likely to complement the limitations of existing Ig-type antibodies, and is particularly likely to exhibit advantages in recognizing antigenic sites that are not recognized due to immune tolerance, or in recognizing glycans or various secondary metabolites.

[0005] Natural forms of VLRs derived from lampreys and hagfish exist in a recombinant form and are fully expressed only in eukaryotic cells such as HEK293 or Sf9 cells; however, expression and purification in prokaryotic cells such as Escherichia coli have been difficult, making it challenging to expand and apply related research. This aligns with the characteristics observed during the recombinant expression of LRR domains in eukaryotes, whereas it contradicts the characteristic of LRR domains in prokaryotes, where large quantities can be produced while maintaining appropriate folding forms and activity during recombinant expression in Escherichia coli. Consequently, it can be inferred that the disulfide bonds primarily located at the amino (N-terminus) and carboxy (C-terminus) regions of the LRR in eukaryotes may be the most significant cause. The inventors have already identified Origami, an Escherichia coli strain that supports disulfide bonds. TM and SHuffle ®Although success was achieved in obtaining a water-soluble form of VLR through the above methods, the production volume and degree of purification did not meet expectations. Meanwhile, there is a successful case of high-volume expression in E. coli by substituting the N-terminus of VLR with the N-terminus of the Internalin B protein found in Listeria monocytogenes; this result supports the hypothesis that the N-terminus of VLR is a determinant distinguishing between expression in prokaryotes and eukaryotes.

[0006] Accordingly, the present invention aimed to develop a method to replace and improve the previously known internalin B among proteins capable of substituting the amino terminus of VLR.

[0007] Meanwhile, Korean registered patent No. 1219628 discloses 'a water-soluble fusion polypeptide in which the N-terminus of an internalin protein and an LRR family protein are fused, and a method for preparing the same', and Korean registered patent No. 1572219 discloses 'a method for improving a lipibody protein including a repeating module', but there is no description of 'a water-soluble fusion polypeptide in which a chimeric peptide derived from an internalin protein is fused to the amino terminus and the use thereof' of the present invention.

[0008] The present invention was derived from the above requirements. The inventors compared and analyzed existing known variable lymphocyte receptors (VLRs) and modified chimeric or leucine-rich repeat (LRR) sequences in three-dimensional structures, fixed them into a structure with eight β-strands, and selected two candidates for the amino-terminal portion based on the stabilization order of internalin proteins according to Instability Index (II) analysis. They then performed a basic verification to determine whether a fusion model could be implemented through modeling using the artificial intelligence protein structure prediction programs Alphafold2 and Chaidiscovery. Afterward, the verified structure was actually expressed and purified in E. coli, and the purity and quantity of the finally obtained purified protein were compared to select the optimal construct. The amino-terminal portion used therein (Sequence No. 1) was named Hyper Bridge to indicate that it can be expressed in both eukaryotic cells and bacteria. In addition, the entire construct including the hyperbridge or the entire construct with some modifications applied thereto was named SB, derived from Scaffold Binder, and was named SB1, SB2, SB3, SB4, and SB5 in the order of modification. SB1 is a sequence consisting of a total of 266 amino acids including the hyperbridge, and the part marked XXX within the sequence indicates that 20 amino acids exist randomly. SB2 was designed to have four lysine (Lys)-glutamic acid (Glu) ionic bond pairs on the side of the scaffold in SB1, SB3 was designed to have four arginine (Arg)-glutamic acid (Glu) ionic bond pairs on the side of the scaffold in SB1, SB4 was designed by substituting the carboxy terminus of SB1 with a sequence derived from ShigellaIpaH7.8 (invasion plasmid antigen H7.8), and SB5 was designed by substituting the 176th to 179th amino acids of SB1 with Xn (X is any amino acid, and n is an integer from 12 to 18).SB1 of the present invention is designed to increase expression and stability compared to existing lipibodies, SB2 and SB3 are designed to increase thermal stability compared to SB1 by adding four ionic bonds, SB4 is designed to significantly increase expression in E. coli by substituting the carboxy terminal portion with a sequence derived from ShigellaIpaH7.8 that lacks a disulfide bond, thereby alleviating the burden of post-translational modification, and SB5 is designed to increase binding strength compared to SB1.

[0009] To solve the above problem, the present invention provides a chimeric peptide in which the amino-terminal sequences of internalin H and internalin B proteins are fused, and a fusion polypeptide in which an LRR (leucine-rich repeat) peptide is fused.

[0010] In addition, the present invention provides a nucleic acid encoding the fusion polypeptide.

[0011] In addition, the present invention provides a recombinant vector comprising the nucleic acid.

[0012] In addition, the present invention provides a host cell transformed with the recombinant vector.

[0013] In addition, the present invention provides a method for producing a fusion polypeptide, comprising the steps of: transforming a host cell with the recombinant vector; and culturing the transformed host cell to recover the fusion polypeptide.

[0014] In addition, the present invention provides a method for selecting variable lymphocyte receptors that bind to a target protein, comprising the steps of: constructing a variable lymphocyte receptor library in vitro or in vitro using the fusion polypeptide; and performing a phage display from the constructed library.

[0015] In addition, the present invention provides a chimeric peptide in which the amino-terminal sequences of each protein of internalin H and internalin B, composed of the amino acid sequence of SEQ ID NO. 1, are fused.

[0016] In addition, the present invention provides a recombinant vector characterized by a polynucleotide encoding the chimeric peptide and a gene encoding a target protein being sequentially linked.

[0017] In addition, the present invention provides a method for increasing the solubility and expression amount of a target protein in vitro or in vitro, comprising the step of transforming a host cell with the recombinant vector to express a target protein-coding gene.

[0018] When using a chimeric peptide in which the amino-terminal sequences of internalin H and internalin B proteins are fused according to the present invention, the protein expression amount of the LRR (leucine-rich repeat) fusion polypeptide increases by more than 2.8 times compared to the conventional technology using only the amino-terminal sequence of internalin B protein, and the screening efficiency of variable lymphocyte receptor (VLR)-based antibodies against target antigens is maximized through the production of a synthetic library using a construct containing the chimeric peptide, thereby allowing for the expectation of cost reduction effects in the development and mass production of VLR as an antibody material.

[0019] Figure 1 shows the amino-terminal sequence information and structures of a total of seven internalin family proteins overlaid for comparison. The inventors substituted the amino-terminal sequence of each internalin protein with the amino-terminal (indicated by the dotted circle) of the VLR scaffold structure (PDB:3RFS), modeled it using AlphaFold2, visualized the structures using the PyMOL program, and then reviewed the validity of the model and the possibility of actual expression.

[0020] Figure 2 shows the results of the analysis of the Instability Index (II) of polypeptides forming the amino-terminal domain of each internalin protein, and it can be seen that the degree of stabilization is in the order of internalin H, internalin A, and internalin B.

[0021] Figure 3 shows the amino acids that maintain the internal backbone and the amino acids that are external but important for maintaining the backbone in the structure constituting the first β-strand of a previously known repebody, and compares the sequences of these parts with the identical parts of internalin H and internalin A.

[0022] Figure 4 shows the three-dimensional structure of a fusion polypeptide modeled after substituting the amino terminus (containing internalin B) of the VLR scaffold structure (PDB:3RFS) with internalin A, internalin H, or a chimeric peptide of internalin H and B.

[0023] Figure 5 shows the results of expressing four fusion polypeptides according to Figure 4 in E. coli and confirming them on an SDS-PAGE gel after gel filtration purification, showing information such as integrity, expression amount, and purity.

[0024] Figure 6 is a quantification of the results of Figure 5, and is the result of comparing the expression levels of a fusion polypeptide in which a portion of the amino-terminal sequence of internalin B protein is fused, with the expression level set to 1X.

[0025] FIG. 7 shows modeling structures of a second structure (SB2) and a third structure (SB3) in which ionic bond pairs are introduced in a first structure (SB1) of a fusion polypeptide containing chimeric peptides of internalin H and B, each implementing ionic-ionic bonding through glutamic acid (Glu) and lysine (Lys) or glutamic acid and arginine (Arg).

[0026] Figure 8 shows the thermal stability of the first structure (SB1), the second structure (SB2), and the third structure (SB3) measured using a Tycho instrument from Nano Temper, and shows that SB2 and SB3, which have ionic bond pairs, have relatively higher thermal stability than SB1.

[0027] Figure 9 shows the modeling structure of the LRR domain of Shigella IpaH7.8 (invasion plasmid antigen H7.8) (top) and the modeling structure of the fourth structure (SB4) in which the carboxy terminus of the first structure (SB1) LRR peptide is substituted with a sequence derived from Shigella IpaH7.8 (bottom).

[0028] Figure 10 shows the results of expressing and purifying SB4 in E. coli, with the left side showing size-exclusion column data and the right side showing SDS-PAGE gel data, which demonstrates the expression amount and purity.

[0029] FIG. 11 shows a modeling structure of a fifth structure (SB5) in which CDR is introduced into a first structure (SB1) of a fusion polypeptide containing chimeric peptides of internalin H and B, and was implemented by substituting the 176th to 179th amino acids with Xn (X is any amino acid and n is an integer from 12 to 18).

[0030] To achieve the objective of the present invention, the present invention provides a chimeric peptide in which the amino-terminal sequences of internalin H and internalin B proteins are fused, and a fusion polypeptide in which an LRR (leucine-rich repeat) peptide is fused.

[0031] In the fusion polypeptide according to the present invention, the chimeric peptide formed by fusing the amino-terminal sequences of the internalin H and internalin B proteins may be composed of the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0032] The chimeric peptide of the present invention is a chimeric peptide in which a portion of the α-helical capping motif and repeat module of internalin H and internalin B, selected through instability index analysis and molecular dynamics simulation, is fused, and performs the function of stabilizing protein folding in a prokaryotic host.

[0033] In the present invention, the term "amino terminus (N-terminus) of an internalin protein" refers to the amino terminus of an internalin protein required for the water-soluble expression of a fusion polypeptide.

[0034] The 45th, 47th, 49th, and 50th positions of the chimeric peptide composed of the amino acid sequence of SEQ ID NO. 1 according to the present invention may be any amino acid, preferably sequentially glutamic acid (Glu), isoleucine (Ile), asparagine (Asn), and asparagine, but are not limited thereto.

[0035] In one embodiment of the present invention, the chimeric peptide composed of the amino acid sequence of SEQ ID NO. 1 is formed by fusing the amino terminal sequence of an internalin H protein composed of the amino acid sequence of SEQ ID NO. 4 and the amino terminal sequence of an internalin B protein composed of the amino acid sequence of SEQ ID NO. 5, wherein the 5th to 43rd amino acids of the amino acid sequence of SEQ ID NO. 1 are derived from the amino terminal sequence of the internalin H protein, and the 1st to 4th amino acids and the 44th to 65th amino acids of the amino acid sequence of SEQ ID NO. 1 are derived from the amino terminal sequence of the internalin B protein.

[0036] In addition, the chimeric peptide according to the present invention may include a chimeric peptide having the amino acid sequence of SEQ ID NO. 1 and a functional equivalent thereof. In the present invention, the term "functional equivalent" refers to a tag that exhibits substantially the same physiological activity as the tag represented by SEQ ID NO. 1, having sequence homology of at least 60%, preferably 80%, more preferably 90%, and even more preferably 95% as a result of the addition, substitution, or deletion of amino acids. "Substantially the same physiological activity" means an activity that increases the water solubility and expression level of a fusion polypeptide formed by fusing the chimeric peptide and the LRR peptide.

[0037] The fusion polypeptide of the present invention may have any one structure selected from the group consisting of: a first structure in which a chimeric peptide having the amino acid sequence of SEQ ID NO. 1 and an LRR peptide having the amino acid sequence of SEQ ID NO. 2 are fused; a second structure in which a lysine (Lys)-glutamic acid (Glu) ionic bond pair is additionally introduced to the LRR peptide of the first structure; a third structure in which an arginine (Arg)-glutamic acid (Glu) ionic bond pair is additionally introduced to the LRR peptide of the first structure; and a fourth structure in which the carboxyl terminus of the LRR peptide of the first structure is substituted with a sequence derived from ShigellaIpaH7.8 (invasion plasmid antigen H7.8); but is not limited thereto.

[0038] In the fusion polypeptide according to the present invention, the addition of a lysine (Lys)-glutamic acid (Glu) ionic bond pair in the second structure may be achieved by substituting the 33rd, 57th, 81st, and 105th amino acids with glutamic acid and substituting the 55th, 79th, 103rd, and 127th amino acids with lysine in an LRR peptide composed of the amino acid sequence of SEQ ID NO. 2; the addition of an arginine (Arg)-glutamic acid (Glu) ionic bond pair in the third structure may be achieved by substituting the 33rd, 57th, 81st, and 105th amino acids with glutamic acid and substituting the 55th, 79th, 103rd, and 127th amino acids with arginine in an LRR peptide composed of the amino acid sequence of SEQ ID NO. 2; and the fourth structure may be achieved by replacing the 152nd to 201st amino acids of the LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with the amino acid sequence of SEQ ID NO. 3. It may be substituted with a sequence derived from ShigellaIpaH7.8, but is not limited thereto.

[0039] In addition, in the fusion polypeptide according to the present invention, the first structure may further substitute the 176th to 179th amino acids of an LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with Xn (where X is any amino acid and n is an integer from 12 to 18); the second structure may substitute the 33rd, 57th, 81st, and 105th amino acids of an LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with glutamic acid, substitute the 55th, 79th, 103rd, and 127th amino acids with lysine, and further substitute the 176th to 179th amino acids of an LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with Xn (where X is any amino acid and n is an integer from 12 to 18); and the third structure may substitute the 33rd, 57th, 81st, and 105th amino acids of an LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with glutamic acid It may be substituted, and the 55th, 79th, 103rd, and 127th amino acids may be substituted with arginine, and additionally the 176th through 179th amino acids in the LRR peptide consisting of the amino acid sequence of SEQ ID NO. 2 may be substituted with Xn (where X is any amino acid and n is an integer from 12 to 18), but is not limited thereto.

[0040] In the present invention, "Xn (where X is any amino acid and n is an integer from 12 to 18)" substituting the 176th to 179th amino acids of the LRR peptide may be derived from the complementarity determining region (CDR) of an existing immunoglobulin antibody or the CDR of a single-domain antibody, and the single-domain antibody may be a variable heavy chain domain of heavy chain (VHH) or a variable new antigen receptor (VNAR), but is not limited thereto.

[0041] In the fusion polypeptide according to the present invention, the LRR peptide may have eight repeat modules consisting of the amino acid sequence XLXLXX [L: leucine, alanine, glycine, isoleucine, or valine, X: any amino acid], but is not limited thereto. The XLXLXX repeat module may be observed in LRR family proteins such as variable lymphocyte repeats.

[0042] In the fusion polypeptide of the present invention, the second and third structures are characterized by increased thermal stability through the addition of ionic bond pairs, which results in a denaturation temperature 3 to 5°C higher than that of the first structure. In the case of the second structure, structural denaturation hardly occurs even at temperatures above 70°C, and in the case of the third structure, it is characterized by being able to provide a stronger positive charge compared to the ionic bond pair of the second structure and exhibiting an electrostatic stabilization effect accordingly. Additionally, the fourth structure is characterized by replacing the carboxyl terminus of the existing LRR peptide (SEQ No. 2) with a sequence derived from Shigella IpaH7.8 that lacks a disulfide bond, thereby increasing the water-soluble expression level of the fusion polypeptide by more than 3 times compared to the first structure when expressed in a prokaryotic host.

[0043] The fusion polypeptide according to the present invention is characterized by increased expression levels and improved structural stability in Escherichia coli compared to a fusion polypeptide in which an LRR peptide is fused with each amino terminus of internalin B, internalin A, or internalin H proteins.

[0044] In addition, the fused polypeptide according to the present invention may have a functional tag additionally fused to an amino terminus or a carboxy terminus, and the functional tag may preferably be selected from a purification tag, a detection tag, an affinity binding tag, an enzymatic conjugation tag, and combinations thereof, but is not limited thereto. More specifically, the functional tag may be a His tag comprising 6 to 12 consecutive histidine residues, an ALFA tag, or an LPXTG sequence for sortase-mediated conjugation (Leucine-Proline-X-Threonine-Glycine, where X is any amino acid, preferably glutamic acid (E)), but is not limited to the above types.

[0045] The above functional tag can be linked to a fusion polypeptide with a flexible linker containing glycine, serine, alanine, or threonine.

[0046] The present invention also provides a nucleic acid encoding the fusion polypeptide and a recombinant vector comprising the nucleic acid.

[0047] The recombinant vector according to the present invention may additionally include a purification tag sequence in addition to the nucleic acid sequence encoding the fusion polypeptide, but is not limited thereto.

[0048] In the present invention, the term "recombination" refers to a cell replicating a heterogeneous nucleic acid, expressing said nucleic acid, or expressing a protein encoded by a peptide, a heterogeneous peptide, or a heterogeneous nucleic acid. A recombinant cell may express a gene or gene fragment not found in the natural form of said cell in either a sense or antisense form. Additionally, a recombinant cell may express a gene found in a cell in its natural state, provided that said gene is modified and reintroduced into the cell by artificial means.

[0049] Additionally, in the present invention, the terms “vector” or “expression vector” refer to a linear or circular DNA molecule comprising a fragment encoding a polypeptide that is linked to function by being composed of elements provided for gene transcription and translation and additional fragments. The additional fragments include a promoter and a transcription termination signal sequence. The vector or expression vector includes one or more replication initiation sites and one or more selection markers, etc. The vector or expression vector is generally derived from plasmid or viral DNA, or includes elements of both. Since plasmids are the most commonly used form of vectors currently, “plasmid” and “vector” are sometimes used interchangeably in this specification.

[0050] Of course, not all vectors function equally in expressing the DNA sequence of the present invention, and likewise, not all hosts function equally for the same expression system. However, those skilled in the art may appropriately select and apply from among various other vectors, expression regulatory sequences, and hosts without departing from the scope of the present invention and without excessive experimental burden. For example, when selecting a vector, the host must be considered because the vector must be replicated within it, and the copy number of the vector, the ability to control the copy number, and the expression of other proteins encoded by said vector, such as antibiotic markers, must also be considered.

[0051] The nucleic acid sequence encoding the fusion polypeptide according to the present invention may use a sequence optimized for the codons of the host cell, but is not limited thereto. In the present invention, the term "codon optimization" refers to changing the codons of a polynucleotide encoding a protein to be preferentially used in a specific organism so that the encoded protein is expressed more efficiently in the organism. Although the genetic code is degenerate in that most amino acids are represented by a few codons referred to as "synonymous" or "synonymous" codons, the codon usage by a specific organism is not random but biased toward specific codon triplets. This bias in codon usage may be higher in relation to specific genes, genes of common function or ancestral origin, highly expressed proteins versus low-copy number proteins, and collective protein-coding regions of the organism's genome.

[0052] The present invention also provides a host cell transformed with the recombinant vector.

[0053] Any host cell known in the art that can stably and continuously clone and express the vector of the present invention in a prokaryotic cell may be used, such as, for example, strains of the genus Bacillus such as E. coli BL21, E. coli JM109, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, and Bacillus churingensis, and intestinal bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.

[0054] In addition, when the vector of the present invention is transformed into a eukaryotic cell, the host cell may be yeast (e.g., Saccharomyce cerevisiae; Pichia pastoris; Kluyveromyces lactis; Kluyveromyces marxianus; Yarrowia lipolytica; Hansenula polymorpha, etc.) or insect cell (e.g., Spodoptera frugiperda Sf9, Sf21, High Five). TM ), animal cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells, etc. may be used.

[0055] The method of transporting the vector of the present invention into a host cell can be carried out by the CaCl2 method, the Hanahan method, and the electroporation method when the host cell is a prokaryotic cell. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by the microinjection method, the calcium phosphate precipitation method, the electroporation method, the liposome-mediated transfection method, the DEAE-dextran treatment method, and the gene bombardment method.

[0056] The present invention also provides a method for producing a fusion polypeptide, comprising the steps of: transforming a host cell with the recombinant vector; and culturing the transformed host cell to recover the fusion polypeptide.

[0057] In the method for producing a fusion polypeptide according to the present invention, the culture of the transformed host cells may be carried out in a medium suitable for the production of the target protein using known techniques. Suitable culture media may be commercially available or prepared according to the ingredients and composition ratios described in publications such as, for example, the catalog of the American Type Culture Collection, but are not limited thereto.

[0058] In addition, in the method for producing a fusion polypeptide according to the present invention, the method for recovering the fusion polypeptide may be recovered from the culture medium by conventional methods including centrifugation, filtration, extraction, spray drying, evaporation, or precipitation, but is not limited thereto.

[0059] In addition, the method for producing a fused polypeptide according to the present invention may further include a step of purifying after recovery. The purification method may include, but is not limited to, various known methods including chromatography (e.g., ion exchange, affinity, hydrophobic and size exclusion), dialysis, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

[0060] The present invention also provides a method for selecting variable lymphocyte receptors that bind to a target protein, comprising the steps of: constructing a variable lymphocyte receptor library in vitro or in vitro using the fusion polypeptide of the present invention; and performing a phage display from the constructed library.

[0061] In the method for selecting a variable lymphocyte receptor according to the present invention, the variable lymphocyte receptor library comprises 20 amino acid variations at the X position of an XLXLXX [L: leucine, alanine, glycine, isoleucine, or valine, X: any amino acid] repeat module within the LRR peptide of the fusion polypeptide.

[0062] In a screening method according to one embodiment of the present invention, a DNA combination using an overlap polymerase chain reaction and an overlap primer (including an NNK degenerate codon) is designed to construct a random library containing the 20 amino acid variations, the designed DNA combination is cloned using a phagemid vector, and the cloned vector is introduced into a bacteriophage to produce a library for phage display.

[0063] In addition, the screening method according to the present invention screened the constructed variable lymphocyte receptor library using a phage display method to screen for variable lymphocyte receptors that specifically bind to a target protein.

[0064] In the present invention, the term "biopanning" refers to a process of selecting only phages from a phage library that displays peptides on their outer walls (coats) that have the property of binding to a target protein (antigen, enzyme, cell surface receptor, etc.) and expressing peptides on their surface.

[0065] In the present invention, the terms "phage" or "bacteriophage" are used interchangeably and may refer to a virus that infects bacteria and replicates within bacteria. The phage or bacteriophage may be used to display a variable lymphocyte receptor (VLR) belonging to the library of the present invention, and may be genetically engineered so that the variable lymphocyte receptor is displayed on the envelope protein of the phage or a fragment thereof. The genetic engineering includes introducing a gene encoding the variable lymphocyte receptor.

[0066] The present invention also provides a chimeric peptide in which the amino-terminal sequences of each protein of internalin H and internalin B, consisting of the amino acid sequence of SEQ ID NO. 1, are fused.

[0067] A chimeric peptide composed of the amino acid sequence of SEQ ID NO. 1 according to the present invention can be used as a fusion tag for the production of recombinant proteins. By using a vector equipped with a functional tag that assists in protein folding or recovery during the production of recombinant proteins, the solubility of the protein can be increased or the ease of the purification process can be improved.

[0068] The present invention also provides a recombinant vector characterized by a polynucleotide encoding the chimeric peptide and a gene encoding a target protein being sequentially linked.

[0069] The above-mentioned recombinant vector may be operably linked to a polynucleotide encoding a chimeric peptide consisting of the amino acid sequence of SEQ ID NO. 1 and a gene encoding a target protein. In the present invention, "operably linked" means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is influenced by the other nucleic acid fragment. That is, the polynucleotide encoding the chimeric peptide consisting of the amino acid sequence of SEQ ID NO. 1 and the gene encoding the target protein may be linked so that their expression can be regulated by a promoter within the vector. The method of linking the promoter, the polynucleotide encoding the chimeric peptide, and the gene encoding the target protein may use conventional techniques that can be easily performed by those skilled in the art, such as PCR, restriction enzyme cleavage, and ligation.

[0070] In the present invention, the term "target protein" refers to any protein that a person skilled in the art intends to produce in large quantities, and which can be expressed in a transformant by inserting a polynucleotide encoding said target protein into a recombinant expression vector. In the recombinant vector of the present invention, said target protein may be a protein having the characteristic of being non-expressible and not expressed solublely in a host cell, and may be a protein such as a toxin, antigen, antibody, or enzyme, but is not limited thereto.

[0071] The present invention also provides a method for increasing the solubility and expression amount of a target protein in vitro or in vitro, comprising the step of transforming a host cell with the recombinant vector to express a target protein-coding gene.

[0072]

[0073] The present invention will be explained in detail below through examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0074]

[0075] Example 1. Chimeric scaffold construct modeling using AlphaFold2

[0076] After replacing the amino-terminal (N-terminal) portion of the existing known VLR scaffold structure (RIPIBOD, PDB:3RFS; https: / www.rcsb.org / structure / 3rfs) with the N-terminal of internalin proteins, modeling was performed using AlphaFold2 (https: / neurosnap.ai / service / AlphaFold2), and the structures were visualized using the PyMOL program. The validity of the model and the potential for actual expression were then examined (Figs. 1 to 3). The modeled structure was superimposed on the existing structure obtained through experiments to confirm that there were no structural defects, and it was examined whether there were any structural trade-offs between the individual amino acid residues in forming the internal structure.

[0077] The DNA sequence of an existing scaffold protein (lipibody, SEQ ID NO. 6) containing internalin B at the N-terminus was synthesized by removing rare codons from E. coli and optimizing them with frequently used codons. This was then cloned into the pET21(+) vector using the CPEC (Circular Polymerase Extension Cloning) method, and a tag sequence containing six consecutive histidine sequences was cloned into the carboxy-terminal (C-terminus) of the protein. Using the above backbone, internalin A, H, or B+H were cloned into the amino-terminals instead of internalin B using the same method (Fig. 4). After transforming the cloned DNA into DH5α cells, the plasmids were extracted and purified to confirm cloning success via Sanger sequencing, and each construct was shuffled again. ® T7 cell lines were transformed. Subsequently, each cell was cultured in 20 ml LB medium for 18 hours, then inoculated into a flask containing 2 L LB medium and OD was set at 37°C. 600 After growing to a value of 0.5, IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added to a concentration of 0.5 mM, and protein expression was induced by incubating at 18°C ​​for 18 hours.

[0078] The cell culture medium in which the above protein expression was induced was centrifuged at 6,000 rpm for 30 minutes, the supernatant was discarded, and the pellet was recovered, resuspended in a buffer containing a protease inhibitor, sonicated, and centrifuged at 20,000 rpm for 1 hour. Afterward, affinity chromatography was performed on the supernatant using nickel resin. SDS-PAGE electrophoresis was performed on the proteins separated and purified by size-exclusion chromatography (Fig. 5), and only the scaffold proteins from the main peak were collected and their respective concentrations were measured.

[0079] Analysis results confirmed that the expression level of a scaffold protein having a chimeric peptide (IntH+IntB) in which a portion of the amino-terminal sequence of the internalin H protein and a portion of the amino-terminal sequence of the internalin B protein are fused was significantly increased compared to a conventional scaffold protein having internalin B (Fig. 6).

[0080]

[0081] Example 2. Expression and purification of a fusion polypeptide (Scaffold Binder 1, SB1) of a chimeric peptide (Hyper Bridge) formed by fusing the amino-terminal sequences of Internalin H and Internalin B proteins with an LRR peptide

[0082] SB1 is a construct in which a chimeric peptide (Hyper Bridge) is located at the amino terminus, in which the amino terminus sequences of internalin H and internalin B proteins are fused, and an LRR peptide is fused to the carboxy terminus of the chimeric peptide. For the expression of SB1, the nucleotide sequences encoding the Hyper Bridge sequence (Sequence No. 1) and the LRR peptide sequence (Sequence No. 2) were each designed as codons optimized for E. coli and synthesized. The LRR peptide sequence (Sequence No. 2) used for the expression experiment is identical to the 66th to 266th amino acids based on the amino acid sequence of Sequence No. 6.

[0083] The synthesized DNA sequence was cloned into a pET21-family vector, and a His tag (6x His) sequence was attached to the carboxy terminus.

[0084] To confirm SB1 expression, transformation was first performed on the Origami™ (Novagen) strain. The Origami™ strain facilitates the formation of disulfide bonds due to the relaxed intracellular reducing environment, but SB1 according to the present invention is designed so that folding occurs stably even without disulfide bonds due to Hyper Bridge.

[0085] After the above transformation, cells were cultured at 37°C using LB medium (or 2xYT medium), and OD 600 When the value reached 0.5, 1 mM IPTG was added, the sample was moved to 18℃ and cultured for 16 hours, and then centrifuged at 6,000 rpm for 30 minutes to recover the cell pellet.

[0086] Harvested cells (wet weight of approximately 2 g per 100 ml of culture medium) were suspended in homogenization buffer (50 mM Tris, 300 mM NaCl, pH 7.5) and sonicated to lyse the cells. After centrifugation, the supernatant was loaded onto a Ni-NTA column and eluted with an imidazole concentration gradient. Size exclusion chromatography (using a Superdex 75 or 200 column) was performed to isolate the monomer peaks of the target protein, and a single band was identified in SDS-PAGE analysis.

[0087] As a result of the analysis, it was confirmed that SB1 of the present invention (corresponding to IntH+IntB in Fig. 6) had an expression level approximately 2.8 times higher than that of the existing repebody (corresponding to IntB only in Fig. 6), and it was found that the protein maintained stable folding in monomeric form (>95% purity).

[0088]

[0089] Example 3. Comparison of thermal stability between SB2 and SB3

[0090] Four lysine (Lys)-glutamic acid (Glu) or arginine (Arg)-glutamic acid (Glu) ionic bond pairs were inserted at specific points among the surface hydrophilic residues on the scaffold side of the SB1 backbone (substituting the 33rd, 57th, 81st, and 105th amino acids with glutamic acid based on the amino acid sequence of SEQ ID NO. 2, and substituting the 55th, 79th, 103rd, and 127th amino acids with lysine or arginine). The construct with the added lysine (Lys)-glutamic acid (Glu) ionic bond pairs was named SB2, and the construct with the added arginine (Arg)-glutamic acid (Glu) ionic bond pairs was named SB3 (Fig. 7). The insertion locations were selected as loop portions between β-sheets, where disruption of the protein backbone is minimized.

[0091] Both the designed SB2 and SB3 were cloned into pET21-family vectors using the same method as SB1 and expressed in E. coli Origami™ strains. The expression and purification processes were similar to those of SB1, and samples with a purity of over 95% were obtained through Ni-NTA and gel filtration steps.

[0092] Intrinsic fluorescence changes were observed over a temperature range (30°C–95°C) under a sample concentration of 0.5 mg / mL using a Tycho instrument from Nanotemper. As a result, as shown in Figure 8, the denaturation temperature (Tm) of SB1 was measured to be around 77°C, SB2 at 83°C, and SB3 at 86°C, confirming the increase in stability due to the introduction of ionic bond pairs. Through this, it was found that the thermal stability of both SB2 and SB3 was improved compared to SB1, and among them, SB3, which introduced an Arg-Glu bond, showed higher stability.

[0093]

[0094] Example 4. Evaluation of C-terminal Substitution and Expression Level of SB4

[0095] SB4 is a construct in which the carboxyl terminus (from the 152nd to the 201st position based on the amino acid sequence of SEQ ID NO. 2) of the SB1 backbone is replaced with the LRR domain sequence derived from ShigellaIpaH7.8 (invasion plasmid antigen H7.8) (SEQ ID NO. 3) (Fig. 9). Since the ShigellaIpaH7.8 domain is known to be free of disulfide bonds, it was predicted that a high yield could be expected even if the refolding process was omitted when expressed in E. coli.

[0096] In the case of SB4, it was determined that no special E. coli cell line was required as the disulfide bond itself is not needed, and SB4 was cloned into a pET21-family vector in the same way as SB1 and expressed in E. coli BL21(DE3).

[0097] SDS-PAGE gel quantification results confirmed that SB4 expressed approximately three times more soluble protein compared to SB1, and the major protein peak was detected during gel filtration. In the case of SB4, the obtained protein concentration was very high, which allowed for the simplification of the concentration process (Fig. 10). Through this, it was confirmed that SB4 is a scaffold form that is highly advantageous for mass production, as it enables high-concentration expression and easy purification.

[0098]

[0099] Example 5. C-terminal substitution of SB5 and evaluation of binding strength

[0100] SB5 is a construct in which the carboxyl terminus of the backbone of SB1 to SB3, that is, the 176th to 179th position based on the amino acid sequence of SEQ ID NO. 2, is substituted with Xn (X is any amino acid and n is an integer from 12 to 18). The above Xn (X is any amino acid and n is an integer from 12 to 18) is a CDR (complementarity-determining region) of an immunoglobulin or nanobody and was used to further enhance the target antigen-specific binding affinity of the fusion polypeptide [fusion of Hyper Bridge sequence (SEQ ID NO. 1) and LRR peptide sequence (SEQ ID NO. 2)] according to the present invention.

[0101] In this embodiment, an SB1 backbone-based fusion polypeptide for a cat antigen (Fel d1) was prepared, and then the 176th to 179th amino acid residues in the SB1 backbone sequence of the fusion polypeptide were replaced with the nanobody-derived CDR sequence EFVASISYGSTTNY (Sequence No. 7) to prepare the fusion polypeptide.

[0102] The designed SB5-based fusion polypeptide was cloned into a pET21-based vector in the same manner as described in Example 2 and expressed in the E. coli Origami™ strain. The expression and purification process was similar to that of SB1, and samples with a purity of over 95% were obtained through Ni-NTA and gel filtration steps. Subsequently, the binding affinity to the feline antigen (Fel d1) was analyzed using biolayer interferometry (BLI) with an Octet device (Octet R8, Sartorius AG), and as disclosed in Table 1 below, it was confirmed that the binding affinity increased.

[0103] Analysis results of antigen binding affinity of SB1 and SB5 KD (M)KD error kd (1 / M·s)Ka error kd (1 / s)kd error SB1 based 114.9 6.9 45x10 -10 1.558x10 5 5.589x10 2 1.790x10 -2 8.712x10 -5 SB5 based 28.382.514x10 -10 1.527x10 5 1.139x10 3 4.334x10 -3 2.071x10 -5 KD (M): Dissociation constantka (1 / M·s): Association rate constantkd (1 / s): Dissociation rate constant

[0104]

[0105] Example 6. Evaluation of the feasibility of constructing a VLRs library using Hyper Bridge-based scaffold proteins

[0106] DNA combinations were designed using polymerase chain reaction (Overlap PCR) and overlap primers (containing NNK degenerate codons) to construct a random library containing 20 amino acid variations for target binding at the XXX positions of the SB1 or SB4 backbone. The DNA combinations were inserted into a phage display vector, such as a phagemid, to attempt phage surface expression in E. coli TG1 strains.

[0107] In the case of SB1 as well, it was confirmed that it is mostly secreted in a water-soluble manner, folds normally on the phage surface, and is functionally expressed, suggesting that it can be applied to future target protein screening. It was inferred that the Hyper Bridge-based scaffold of the present invention is more expressive than existing repibodies, making it easier to construct large-scale libraries and perform iterative screening.

Claims

1. A fusion polypeptide formed by fusing a chimeric peptide in which the amino-terminal sequences of internalin H and internalin B proteins are fused, and an LRR (leucine-rich repeat) peptide is fused.

2. The fusion polypeptide according to claim 1, characterized in that the chimeric peptide formed by fusing the respective amino-terminal sequences of the internalin H and internalin B proteins consists of the amino acid sequence of SEQ ID NO.

1.

3. The fusion polypeptide according to claim 1, wherein the fusion polypeptide has any one structure selected from the group consisting of: a first structure in which a chimeric peptide formed by fusing the amino-terminal sequences of internalin H and internalin B proteins is fused with an LRR peptide formed by the amino acid sequence of SEQ ID NO. 2; a second structure in which a lysine (Lys)-glutamic acid (Glu) ionic bond pair is additionally introduced to the LRR peptide of the first structure; a third structure in which an arginine (Arg)-glutamic acid (Glu) ionic bond pair is additionally introduced to the LRR peptide of the first structure; and a fourth structure in which the carboxyl terminus of the LRR peptide of the first structure is substituted with a sequence derived from ShigellaIpaH7.8 (invasion plasmid antigen H7.8).

4. A fusion polypeptide in which, in paragraph 3, the addition of a lysine (Lys)-glutamic acid (Glu) ionic bond pair of the second structure is obtained by substituting the 33rd, 57th, 81st, and 105th amino acids with glutamic acid and substituting the 55th, 79th, 103rd, and 127th amino acids with lysine in an LRR peptide consisting of the amino acid sequence of SEQ ID NO.

2.

5. In paragraph 3, the addition of the arginine (Arg)-glutamic acid (Glu) ionic bond pair of the third structure is a fusion polypeptide in which the 33rd, 57th, 81st, and 105th amino acids of the LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 are substituted with glutamic acid, and the 55th, 79th, 103rd, and 127th amino acids are substituted with arginine.

6. In paragraph 3, the above-mentioned fourth structure is a fusion polypeptide in which the 152nd to 201st amino acids of an LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 are substituted with a sequence derived from ShigellaIpaH7.8 composed of the amino acid sequence of SEQ ID NO.

3.

7. A fusion polypeptide according to claim 3, wherein the first structure is further characterized by substituting the 176th to 179th amino acids of an LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with Xn (where X is any amino acid and n is an integer from 12 to 18).

8. A fusion polypeptide according to claim 4, wherein the second structure is characterized by substituting the 33rd, 57th, 81st, and 105th amino acids of the LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with glutamic acid, substituting the 55th, 79th, 103rd, and 127th amino acids with lysine, and substituting the 176th to 179th amino acids of the LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with Xn (where X is any amino acid and n is an integer from 12 to 18).

9. A fusion polypeptide according to claim 5, wherein the third structure is characterized by substituting the 33rd, 57th, 81st, and 105th amino acids of the LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with glutamic acid, substituting the 55th, 79th, 103rd, and 127th amino acids with arginine, and substituting the 176th to 179th amino acids of the LRR peptide composed of the amino acid sequence of SEQ ID NO. 2 with Xn (where X is any amino acid and n is an integer from 12 to 18).

10. The fusion polypeptide according to claim 1, characterized in that the fusion polypeptide has increased expression levels and improved structural stability in Escherichia coli.

11. The fusion polypeptide according to claim 1, characterized in that a functional tag is additionally fused to an amino terminus or a carboxy terminus.

12. A fusion polypeptide according to claim 11, wherein the functional tag is selected from a purification tag, a detection tag, an affinity binding tag, an enzymatic conjugation tag, and combinations thereof.

13. A nucleic acid encoding the fusion polypeptide of any one of claims 1 to 12.

14. A recombinant vector containing the nucleic acid of paragraph 13.

15. Host cells transformed with a recombinant vector according to paragraph 14.

16. A step of transforming a host cell with a recombinant vector according to claim 14; and A method for producing a fusion polypeptide comprising the step of culturing the transformed host cell to recover the fusion polypeptide.

17. A step of constructing a variable lymphocyte receptor library in vitro or in vitro using the fusion polypeptide of any one of claims 1 to 12; and A method for selecting variable lymphocyte receptors that bind to a target protein, comprising the step of performing a phage display from the above-described library.

18. A chimeric peptide formed by fusing the amino-terminal sequences of internalin H and internalin B proteins, which consist of the amino acid sequence of SEQ ID NO.

1.

19. A recombinant vector characterized by sequentially linking a polynucleotide encoding the chimeric peptide of claim 18 and a gene encoding a target protein.

20. A recombinant vector according to claim 19, characterized in that the target protein is a non-expressing protein.

21. A method for increasing the solubility and expression amount of a target protein in vitro or in vitro, comprising the step of transforming a host cell with the recombinant vector of claim 19 to express a target protein-coding gene.

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