KR100829727B1 - Methods for Increasing Solubility of Recombinant Proteins or Peptides - Google Patents

Abstract

The present invention includes a vector comprising (i) a promoter, (ii) Plasmid Acromobacter Secretion (PAS) factor-coding nucleotide sequence operably linked to said promoter, and (iii) a recombinant protein or peptide-coding nucleotide sequence, The PAS factor and the recombinant protein or peptide are expressed in the form of a fusion protein, and the recombinant protein or peptide in the form of the expressed fusion protein is characterized in that the solubility is increased compared to the recombinant protein or peptide to which the PAS factor is not bound. Or it relates to a composition for enhancing the solubility of the peptide, and a method for increasing the solubility of the recombinant protein or peptide using the same. According to the present invention, it is possible to obtain a recombinant protein or peptide which has not been efficiently obtained through a cloning process due to its solubility in the conventional expression system.

Protein, peptide, solubility, expression, PAS

Description

Method for Increasing Solubility of Recombinant Proteins or Peptides

1A-1E are genes of specific vectors used in the present invention. P _T7 , MCS, km and lacI represent T7 promoter, multiple cloning site, kanamycin resistance gene and LacI coding sequence, respectively. His-tag, Trx-tag, Nus-tag and GST-tag represent 6 x His (hexahistidine), Trx (Thioredoxin), Nus (N utilization substance A) and glutathione S-transferase genes, respectively.

Figure 2 is a graph comparing the expression of the expression of the total of 50 prokaryotic and eukaryotic proteins using a conventional vector and the PAS fusion vector of the present invention. Con> PAS: When the expression vector of the conventional vector is more, Con = PAS: When the expression amount of the conventional vector and the PAS vector is the same, Con <PAS: When the expression of the PAS vector is more, Con X PAS: The conventional vector and the PAS No expression in both vectors.

Figure 3 is a photograph showing the results of electrophoresis on the representative proteins of prokaryotes and eukaryotes successful expression using the PAS fusion vector of the present invention. Glur 1, a glutamate receptor 1 which is a type of membrane protein related to human neurotransmission; HP1496, Helicobacter pylori ) HP1496 protein, a type of protein. The numbers in parentheses next to Glur 1 indicate the order in which the sense primers and antisense primers used in the polymerase chain reaction are attached to the DNA template. Lanes 1-5 correspond to molecular weight markers, before induction of conventional vector expression, after conventional vector expression induction, before PAS fusion vector expression induction, and after PAS fusion vector expression induction.

4A is a gel photograph of solubility analysis when HP (Hypothetical protein) 0175, a prokaryotic protein, is expressed using a conventional N-His-Tag vector and a PAS-N-His-Tag vector of the present invention.

Figure 4b is the solubility was analyzed in the case of expressing the ubiquitin-binding enzyme protein (Ubiquitin-conjugation enzyme protein, Ku007066) of the eukaryotic protein using the conventional Nus-Tag vector and the PAS-Nus-Tag vector of the present invention Gel picture. 4A and 4B, lanes 1-5 correspond to molecular weight markers, cells before expression induction, cells after expression induction, supernatants after cell disruption, and cell pellets after cell disruption.

The present invention relates to a method for increasing the solubility of a recombinant protein or peptide, and more particularly, to a method for increasing solubility in mass production of a recombinant protein or peptide through a DNA cloning process using a vector.

One of the main reasons for cloning useful genes in biotechnology is to overexpress useful genes in specific microorganisms to mass produce useful proteins. It is difficult to accurately predict the market size of protein products in the bio-industrial market, but it is at the core of the overall market size and accounts for around 60%. In the future, the size of the protein market is expected to increase rapidly. In particular, the sales area is expanding due to the expansion of product use, and the market can be expanded due to the production of improved products, and through the discovery and production of new and new products. It is expected to generate demand.

In order to develop recombinant microorganisms that produce useful proteins at an industrial level, the focus of research has been mainly on the development of recombinant hosts for stable production of foreign proteins and the development of expression vectors for secreting / expressing foreign genes with high efficiency. .

However, a large number of proteins may not be expressed to the desired level, or even if they are expressed, they may not fold properly, resulting in agglutination of inclusion bodies, which can lead to useless forms that lose their function.

On the other hand, Vibrio sepsis is infected through the reproduction of fish and shellfish contaminated with Vibrio vulnificus or through wounded skin by seawater contaminated with bacteria. Vibrio sepsis has a short incubation period and, once developed, progresses rapidly and misses the timing of effective antimicrobial treatment. The mortality rate from this disease (40-50%) is very high, requiring early diagnosis and prompt treatment. In addition, in Japan and Korea, where fish and shellfish are commonly reproduced, it frequently occurs during summer when seawater temperature increases rapidly, but clinical cases are continuously reported all over the world.

Vibrio vulnificus , the causative agent of Vibrio sepsis, is a Gram-negative bacterium that lives in the sea and grows well in NaCl concentrations of 1-3% dls. It was long termed a different bacterium and was found to break down lactose unlike V. parahemolyticus and was named V. vulnificus in 1979. The bacterium is colistin resistant, but susceptible to ampicillin and carbenicillin to distinguish it from other similar bacteria. It is usually a curved shoot and the cells are surrounded by capsules. Vibrio sepsis with capsular material is a measure of the virulence of bacteria because it resists the bactericidal action of serum and has anti-phagy, high lethality and strong tissue penetration. Vibrio vulnificus produced by vibrio vulnificus is directly involved in causing or causing symptoms of the three major types-bactericidal, enzymes and other factors. These virulence factors are overexpressed to survive rapid environmental changes after Vibrio sepsis is infected with humans.

In order to infiltrate the human body, Vibrio septic bacterium ( V. vulnificus ) is to express a variety of new toxic factors, PAS (Plasmid Acromobacter Secretion) factor is one of them is a small protein consisting of 76 amino acids. When Vibrio vulnificus is present in the natural environment, the PAS factor is not expressed. When the human body is infected, excess PAS factor is expressed.

PAS factor was not expressed in Vibrio sepsis grown in vitro, but was expressed in vitro only when the PAS factor gene was cloned into the plasmid and overexpressed in Vibrio sepsis and most of the expressed PAS factor was observed only in the periplasmic fraction. (Tokugawa, K., et al., J. Biotechnol . , 35: 69-76 (1994); Tokukawa, K. et al., J. Biotechnol . 37: 33-37 (1994)).

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made efforts to overcome the technical limitation that the solubility of the conventional expression system is very low so that the recombinant protein or peptide cannot be efficiently obtained. As a result, when the expression of the protein or peptide is expressed by fusing PAS (Plasmid Acromobacter Secretion) factor, the solubility is increased. By discovering that this can be greatly increased, the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for enhancing solubility of a recombinant protein or peptide.

Another object of the invention relates to a method of increasing the solubility of a recombinant protein or peptide.

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

According to one aspect of the present invention, the present invention provides a kit comprising: (i) a promoter, (ii) a Plasmid Acromobacter Secretion (PAS) factor-coding nucleotide sequence operably linked to the promoter and (i) a recombinant protein or peptide-coding nucleotide sequence The vector includes a vector, wherein the PAS factor and the recombinant protein or peptide is expressed in the form of a fusion protein, and the expressed protein or peptide in the form of the fusion protein has a solubility in comparison with a recombinant protein or peptide having no PAS factor bound thereto. It provides a composition for enhancing solubility of a recombinant protein or peptide, characterized in that increased.

According to another aspect of the present invention, the present invention provides a method of increasing the solubility of a recombinant protein or peptide comprising the following steps: (a) (i) a promoter, (ii) PAS operably bound to said promoter Acromobacter Secretion) transforming a host cell with a factor-coding nucleotide sequence and (iii) a vector comprising said recombinant protein or peptide-coding nucleotide sequence, wherein said PAS factor and recombinant protein or peptide are expressed in the form of a fusion protein. Become; (b) culturing the transformed host cell and expressing the recombinant protein or peptide; (c) crushing the host cell to obtain a cell lysate; And (d) obtaining the recombinant protein or peptide lysed from the cell lysate, wherein the obtained recombinant protein or peptide is in fused form with PAS factor and the recombinant protein or peptide in fusion protein form is PAS factor. Solubility is increased compared to recombinant proteins or peptides that do not bind.

The present inventors have tried to overcome the technical limitation that the solubility in the conventional expression system is very low so that a recombinant protein or peptide cannot be efficiently obtained. As a result, it has been found that the solubility can be greatly increased by expressing proteins or peptides by fusion of Plasmid Acromobacter Secretion (PAS) factor.

In the present specification, the term “solubility” refers to a protein or peptide expressed in a transformant and the desired protein or peptide is dissolved in a supernatant when the lysate obtained by crushing the transformant is centrifuged. The undissolved ones are in the pellet when the crushed liquid is centrifuged.

The present inventors first isolated the PAS factor, which is a specific secretory promoting factor in vivo from Vibrio sepsis, and manufactured a PAS fusion vector containing the isolated PAS factor (see Korean Patent Application Publication No. 2006-0006204). . However, the main content of this patent document is the expression of recombinant proteins, and studies of solubility have not been disclosed, taught or implied.

Solubility is a very important factor in obtaining a recombinant protein or peptide through cloning. Expression is also an important factor in obtaining recombinant proteins or peptides, but high solubility is also an important factor in obtaining recombinant proteins or peptides efficiently.

Vectors used in the present invention can be constructed through a variety of methods known in the art, specific methods thereof are described in Sambrook et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

As used herein, the term “operatively linked to” refers to nucleic acid expression control sequences (eg, promoters, signal sequences, or arrays of transcriptional regulator binding sites) and other nucleic acid sequences (eg, PAS factor-coding sequences). Functional binding between), whereby the regulatory sequence controls the transcriptional and / or translational process of the other nucleic acid sequence.

Vectors of the invention can typically be constructed as a vector for expression. Preferably, the vector of the present invention is a vector for expressing a recombinant peptide or protein. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, the tac promoter, lac) that can promote transcription Promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac 5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding for initiation of detoxification It is common to include site and transcription / detox termination sequences. If E. coli is used as the host cell, E. coli Promoter and operator sites of the tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . , 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., . Ann Rev Genet, 14:. . 399-445 (1980)) may be used as a control region.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phages (eg, λgt4? ΛB, λ-Charon, etc., which are often used in the art. , λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the genome of the mammalian cell (for example, metallothionine promoter) or a promoter derived from a mammalian virus (for example, adeno) virus late promoter, the vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and HSV tk Promoter) can be used and generally has a polyadenylation sequence as a transcription termination sequence.

The vector used in the present invention is a glutathione S-transferase (GST) at a position adjacent to the 5'- or 3'-terminus of the PAS factor-encoding nucleotide sequence to facilitate purification of the recombinant peptide or protein expressed therefrom, Nucleotide sequences encoding maltose binding protein (MBP), FLAG, 6x His (hexahistidine), NusA (N utilization substance A) or Trx (Thioredoxin) are additionally included. Because of the additional sequence for this purification, the protein expressed in the host is purified quickly and easily through affinity chromatography.

According to a preferred embodiment of the present invention, the fusion protein expressed by the vector containing the fusion sequence is purified by affinity chromatography. For example, if glutathione-S-transferase is fused, glutathione, which is a substrate of this enzyme, can be used, and if 6x His is used, a Ni-NTA His-binding resin column can be used to rapidly remove the desired recombinant peptide or protein. Can be obtained easily.

Meanwhile, the vector of the present invention includes an antibiotic resistance gene commonly used in the art as an optional marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.

According to a preferred embodiment of the present invention, the 5'-end of the recombinant protein or peptide-coding nucleotide sequence in the vector used in the present invention further includes a protease recognition sequence-coding nucleotide sequence. More preferably, the protease recognition sequence-coding nucleotide sequence is a tobacco etch virus protease (TEV) recognition site-coding nucleotide sequence, most preferably the nucleotide sequence described in SEQ ID NO: 3. Fusion proteins of expressed recombinant peptides or proteins can be obtained by isolation from other sequences by treatment of proteolytic enzymes.

According to a preferred embodiment of the present invention, the vector has a genetic map of any one of FIGS. 1A to 1E. Most preferably, the vector used in the present invention comprises a tobacco etch virus protease (TEV) recognition site-coding nucleotide sequence at the 5'-end of the recombinant protein or peptide-coding nucleotide sequence in the genetic map of FIGS. 1A-1E. do.

The protein or peptide-coding sequence included in the vector of the present invention is not particularly limited, and hormones, hormone analogs, enzymes, inhibitors, signaling proteins or portions thereof, antibodies or portions thereof, single chain antibodies, binding proteins or binding thereof Domains, antigens, adhesion proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators, blood coagulation factors and nucleotide sequences encoding plant biodefense inducing proteins. The peptide-coding sequence included in the vector of the present invention is not particularly limited, and is preferably a nucleotide sequence encoding bioactive fragments of wild-type protein.

More preferably, the recombinant protein or peptide-coding sequence included in the vector used in the present invention may include Helicobacter pylori-derived Hp0096, Hp0331, Hp0572, Hp0844, Hp1069 and Hp1355 proteins; And human-derived Ku0012914, Ku13377, Ku001813, Ku001951, Ku007066, Ku002463, Ku002513, Ku005888 and Ku009023, most preferably Helicobacter pylori-derived Hpotetical protein (Hp) 0175 or human-derived ubiquitin binding enzyme (Ubiquitin-conjugation enzyme) Ku007066.

Host cells capable of stable and continuous cloning and expression of vectors in the methods of the invention are known in the art and can be used with any host cell, for example E. coli JM109, E. coli BL21 (DE3), E. coli Bacillus genus strains such as DH5α, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Cera Enterobacteria and strains, such as Tia Marsesons and various Pseudomonas species.

In the case of transforming a vector of the present invention into a eukaryotic cell, as a host cell, yeast ( Saccharomyce) cerevisiae ), insect cells and human cells (eg, CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and the like.

The method of carrying the vector of the present invention into the host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9: 2110-2114 (1973), when the host cell is a prokaryotic cell . ), One method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9: 2110-2114 (1973); and Hanahan, D., J. Mol . Biol . , 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic . Acids Res . , 16: 6127-6145 (1988)). In addition, when the host cell is a eukaryotic cell, fine injection method (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology , 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene , 10:87 (1980)), DEAE- Dextran Treatment (Gopal, Mol . Cell Biol . , 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc . Natl . Acad . Sci . , 87: 9568-9572 (1990)) and the like can be injected into host cells. have.

Vectors injected into a host cell can be expressed in the host cell, in which case a large amount of recombinant peptide or protein is obtained. For example, when the expression vector contains a lac promoter, the host cell may be treated with IPTG to induce gene expression.

According to a preferred embodiment of the present invention, the host cell used in the present invention is E. coli , most preferably E. coli BL21 (DE3).

In the method of the present invention, the culture of the transformed host cell can be carried out using a medium commonly used in the art. For example, when the transformant is a prokaryotic cell (eg, E. coli ), the transformant may be cultured using LB (Luria-Bertani) medium. When the transformant is an animal cell, for example, the transformant may be cultured using Eagle's MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)).

Various methods of culturing the transformants are well known to those skilled in the art, see Sambrook et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

In the method of the present invention, the method of disrupting the cultured host cell is a method known in the art, such as hand homogenizer, mincing, grinding, blade homogenizer, French press, ultrasonic grinding, beads This can be done using a wheat or Mannton-Gaulin homogenizer (Robert K. Scopes, Protein Purification , 2nd ed. Springer-Verlag New York Inc. (1988)).

The cell disruption solution thus obtained contains cell debris and lysed substances. According to the method of the present invention, the cell lysate contains a large amount of the recombinant protein or peptide of interest in a dissolved state. Centrifugation of cell lysate involves a large amount of recombinant protein or peptide in the supernatant. The recombinant protein or peptide in the supernatant has a form in which the PAS factor is fused, and the recombinant protein or peptide in the fusion protein form has increased solubility compared to the recombinant protein or peptide to which the PAS factor is not bound.

Obtaining the lysed recombinant protein or peptide from the cell disruption solution may be performed during cell culture in a continuous culture method or after cell culture is terminated in a batch culture method.

According to a preferred embodiment of the invention, the separation of the recombinant peptide or protein is carried out by recovering the fusion protein in purified form. For example, recombinant peptides or proteins can be separated using ammonium sulphate precipitation, ultrafiltration membranes with constant molecular weight cut-off values and / or column chromatography (size, charge, hydrophobicity or affinity using cell disruption). Obtained in purified form).

According to a preferred embodiment of the invention, the obtaining of the recombinant peptide or protein is carried out using affinity chromatography. For example, using a resin column bound to glutathione when the recombinant peptide or protein is fused to GST, and a Ni-NTA His-binding resin column when fused to 6x His, the desired recombinant peptide or protein can be quickly and easily. You can get it.

According to the present invention, it is possible to obtain a recombinant protein or peptide which has not been efficiently obtained through a cloning process due to its solubility in the conventional expression system.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Strains and Plasmids Used

The properties of the strains and plasmids used in the examples are summarized in Table 1.

Strain  Characteristic source Esherichia coli BL21 (DE3) F- ompT hsdS (rB - mB -) gal dcm (DE3) Novagen DH5α supE44 , DlacU169 ( f80lacZDM15 ), rec A1 , endA 1 , thi- 1 , gyr A96 , rel A1  Plasmid Characteristic pET30a His-tag expression vector, Kan ^R pET29a His-tag expression vector, Kan ^R pET28a His-tag expression vector, Kan ^R pET41a GST-tag expression vector, Kan ^R

E. coli strains used in the examples were cultured in LB (Luria Bertani) medium (Merk). Strains were added to 20% glycerol after incubation and stored in -70 ℃ cryogenic freezer.

Example  1: vibrio From sepsis PAS  Factor Separation

After culturing Vibrio vulnificus in 2.5% NaCl heart infusion (HI) medium, sodium dodecyl sulfate (SDS) -protease method (Little, PFR (1987) DNA Cloning: A practical approach. IRL press 3: 19-42) purified the genome DNA of Vibrio bacteria.

Genome DNA was digested with restriction enzyme Sau 3AI and the cut DNA fragments were isolated using agarose gel (Amersham). Agarose gel containing DNA fragments between 0.5 and 1.0 kb was treated with β- agarase (New England Biolabs; NEB) and between 0.5 and 1.0 kb from agarose gels using the QIAEX II gel extraction kit (Qiagen). DNA fragments were obtained. Insert DNA fragments into the pET30a vector (Novagen) and E. coli It was transformed in DH5 α, followed by separating a plasmid DNA library from each of the colonies generated. The isolated plasmid library DNA was transformed and expressed in E. coli BL21 (DE3) (Novagen).

Antibodies that specifically bind to Vibrio bacteria were prepared using the Vibrio bacteria proteins expressed in plasma and BL21 (DE3) of Vibrio sepsis patients, and colony Western blotting analysis was performed using the prepared antibodies and colonies. The prepared expression library was serially diluted and plated at about 500 colonies per plate medium and incubated overnight. Using a sterile velvet cloth, replica plates were incubated in isopropyl-β-D-thiogalactoside (IPTG) containing medium and incubated for 5 hours to induce antigen expression from cloned genes. It was. The bacterial culture plate was exposed to chloroform vapor for 15 minutes to expose the protein expressed by lysing bacteria, and then the nitrocellulose filter paper was superimposed on this for 15 minutes to transfer the protein. The protein-transferred filter paper was blocked with 5% skim milk powder, 0.5% Tween 20, pH 7.2 phosphate buffered saline overnight at 4 ° C. in a refrigerator, and then diluted with recovery antibody sera of at least 1: 5000 for 1 hour at room temperature. Reacted for a while. The peroxidase-conjugated anti-human antibody was reacted as a secondary antibody and then determined by ECL chemiluminescence kit (Amersham Co.) and 12 clones showing reactive colonies were selected.

Subsequently, the 12 colonies were analyzed for DNA sequences using an ABI Prism 377 automatic DNA sequencer (Perkin-Elmer Applied Biosystems).

Among the various antigen genes obtained through DNA sequence analysis, PAS factor genes estimated to be protein secretion factors were identified and secured, and DNA sequencing results of the obtained PAS factor genes are the same as in the sequence listing No. 1 sequence.

Example  2: PAS  Expression Vector Preparation Using Factors

Using the PAS factor cloned in the pET30a vector as a template, the PAS factor (231 bp) containing the restriction enzyme Nde I recognition site at both ends of the DNA was amplified. The sense primer used for the amplification was 5'-GGT CATATG AAAGCCCTC (underlined is Nde I recognition site) and the antisense primer was 5'-GCC CATATG GTGTTCAGG. PCR-amplification was performed using 100 ng of template, 0.2 pM primer, 0.25 mM dNTPs, and 2 units of pfu DNA polymerase (Stratagene). Polymerase chain reaction conditions were 5 minutes denaturation at 94 ℃; 25 cycles of reaction at 94 DEG C for 30 seconds, 50 DEG C for 30 seconds and 72 DEG C for 1 minute; Finally it is extended for 10 minutes at 72 ° C. The PAS factor of 231 bp amplified by the polymerase chain reaction was purified using a QIAEX II gel extraction kit (Qiagen).

Trx-Tag coding sequence and TEV (Trx-Tag coding sequence upstream of His-Tag (pET28a, Novagen), Trx-Tag [pET28a vector (Novagen) to be used for the development of PAS fusion vectors among amplified PAS factor genes and commercially available vectors) vector inserting the tobacco etch virus protease recognition site], Nus-Tag [the vector inserting the NusA-Tag coding sequence and the TEV (tobacco etch virus protease) recognition site upstream of the multiple cloning position of the pET28a vector (Novagen)] and GST -Tag vector (pET41a, Novagen) was purified after treatment with restriction enzyme Nde I (NEB). The purified PAS factor gene was cloned into each vector to develop a new expression vector, PAS fusion vector. PAS fusion vectors are classified into PAS-N-His-Tag vector, PAS-C-His-Tag vector, PAS-Trx-Tag vector, PAS-Nus-Tag vector and PAS-GST-Tag vector, depending on the type and location of the tag. Named. The PAS-N-His-Tag vector and the PAS-C-His-Tag vector inserted 6x His Tag before and after the PAS factor based on the pET28a plasmid, followed by the tobacco etch virus protease (TEV), one of the proteolytic enzymes. A recognition site (GAA AAC CTG TAT TTT CAG GGT) and multiple cloning sites (MCS) can be inserted to clone external DNA using a variety of restriction enzymes. The PAS-Trx-Tag vector, the PAS-Nus-Tag vector, and the PAS-GST-Tag vector insert the Trx Tag, Nus Tag, and GST Tag after the PAS factor, respectively, and then the TEV (tobacco etch virus protease) recognition site and MCS Is the vector that inserted.

Gene maps of the developed PAS fusion vectors PAS-N-His-Tag vector, PAS-C-His-Tag vector, PAS-Trx-Tag vector, PAS-Nus-Tag vector and PAS-GST-Tag vector are shown in FIG. It is shown at -1e.

Example  3: of the present invention PAS  Performance Analysis of Fusion Vectors

To verify the performance of the five PAS fusion vector systems developed, expression and solubility experiments were performed on human and bacterial proteins.

Proteins used to verify expression performance were not expressed in existing expression vector systems on the market or were very low in solubility. As prokaryotic proteins, 16 Helicobacter pylori- derived proteins (HP0331, HP0096, HP0175, etc.) This was used. Eukaryotic proteins include six muscle proteins related to muscle relaxation / contraction (RyR 1-6; Ryanodine receptor 1-6) and six human neurotransmission membrane proteins (GluR 1-6; Glutamate receptor 1-6) and 22 other human-derived proteins (Ku001813, Ku002463 ... C17, hs43125, Q13510, etc.) were used for performance verification.

Prokaryotic Proteins Used for Expression Verification Kinds Protein name Characteristic Prokaryotic cells HP0096 Phosphoglycerate dehydrogenase HP0175 Hypothetial protein Hp0331 Septum site - determining protein min  D HP0332 Cell division topologlcal spcificity factor HP0361 tRNA pseudouridine synthase  A HP0572 Adenine phosphoribosyltransferase HP0844 Phosphomethylprimidine kinase HP0925 Recombination protein recR HP1053 septum site - determining protein min  C HP1069 - ATP cell division protein ftsh homolog protein HP1069 - full cell division protein ftsh homolog protein HP1069 - Protease cell division protein ftsh homolog protein HP1178 purine nucleoside phosphorylase deo  D- type HP1337 nicotinate - nucleotide adenyltransferase HP1355 nicotinate - nucleotide pyrophosphorylase HP1496 50 s ribosomal protein L25

Eukaryotic Proteins Used to Validate Expression Kinds Protein name Characteristic Eukaryotic cells Ku001813 ARD I homolog A. Ku001951 Calcyclin binding protein Ku002463 Protein phosphates 2A isoform 1 Ku002513 38 kDa MOV34 Isologue Ku003713 PTD015 protein Ku003880 (1-197) calcipressin Ku003880 (26-197) calcipressin Ku003880 (88-197) calcipressin Ku005888 CT11-human protein C20orf11 Ku007066 ubiquitin-conjugation enzyme E2 Ku009023 (1-228) CWF 19-like I protein Ku009023 (328-474) CWF 19-like I protein Ku009460 Importin-alpha re-exporter Ku012914 NDRG2 protein Ku013377 resisrance to inhibitors of cholinesterase 8 Ku015323 Homeobox protein CDX-2 Ku026621 Hypothetial protein HH114 C17  (1M + 139S) Cartilage Promoting Related Protein C17  (29T + 139S) Cartilage Promoting Related Protein hs43125 esophageal cancer realated gene  4- protein Q13510 Acid ceramidase Q15274 Nad  C GluR  1 (1,1) Human neurotransmission Membrane protein Glutamate receptor GluR  1 (3,3) GluR  1 (3,4) GluR  2 (1,1) GluR  2 (2,2) GluR  2 (2,3) RyR1 Muscle relaxation, contraction Membrane protein Ryanodine receptor RyR2 RyR3 RyR4 RyR5 RyR6

First, genes of commercially available conventional vectors, the invented PAS fusion vector, and target proteins to be used for expression experiments were cut with restriction enzymes Hind III and Xho I (NEB), and the target protein genes were cloned into each vector. The cloned subject protein genes were transformed into E. coli BL21 (DE3), and the transformed colonies were inoculated in 3 ml liquid medium containing kanamycin at a concentration of 25 μg / ml and cultured in a 37 ° C. incubator for at least 16 hours. A portion (500 μl) of the culture was reinoculated into 50 ml liquid medium and incubated at 37 rpm at 37 rpm, and IPTG (0.25 mM) was added when the absorbance of the culture was 0.45 at a wavelength of 600 nm using a spectrophotometer. It was. Cells were incubated for 6 hours after IPTG addition and cells were collected using a centrifuge (3,000 rpm, 10 minutes, 4 ° C.). 12% acrylamide gel electrophoresis was performed with the cell lysate of the collected cells to confirm the expression level of the protein.

As a result of statistically analyzing the expression results of the total proteins used, the percentage of the total protein number to the high expression protein number was higher in the PAS fusion vector than the conventional commercial vector (Fig. 2). 35% of prokaryotic proteins and 46.9% of eukaryotic proteins, including RyR group proteins, which are cell membrane proteins, were not expressed in both the PAS vector system and conventional vector systems. In the N-His-Tag vector, 14 proteins including Ku15323 in eukaryotic cells and four proteins including Hp0332 in prokaryotic cells were not expressed. In the case of C-His-Tag, 16 proteins including Ku3713 in eukaryotic cells and four proteins including Hp0361 in prokaryotic cells were similarly not expressed in all vectors. In the case of Trx-Tag, 33 species including eukaryotic Glur 1 and 15 species including Hp0096 of prokaryotic cells were not expressed. Nus-Tag and GST-Tag vectors showed the same results with 9 proteins of eukaryotic cells and 3 proteins of prokaryotic cells.

In the case of eukaryotic proteins, the expression rate of the PAS vector system was 27.4% higher than that of the conventional vector. Specifically, in the PAS-N-His-Tag vector, GluR1 (1,1), GluR1 (2,2), GluR1 (3,3), GluR1 (3,4), Ku0012914, Ku13377, Ku001813, Ku001951, Ku002463, Ku002513 , Ku005888, Ku009023 (1-228), Ku009023 (328-474), Q13510 and RyR 6; In PASC-His-Tag vectors, GluR1 (1,1), GluR1 (2,2), GluR1 (3,3), GluR1 (3,4), GluR2 (1,1), GluR2 (2,3), Ku0012914 , Ku13377, Ku001813, Ku001951, Ku002513, Ku005888, Ku009023 (1-228), Q15274 and RyR 6; C17 (29T + 139s), Ku12914, Ku001813, Ku001951, Ku002463, Ku003880 (88-197) and Q15274 in the PAS-Nus-Tag vector; In the PAS-GST-Tag vector, C17 (29T + 139s), GluR1 (1,1), GluR2 (2,3), Hs43125, Ku001813, Ku009023 (1-228), Ku009023 (328-474), Ku005888, Ku009023 ( 1-228) and RyR 5; In the PAS-Trx-Tag vector, Q15274 showed more expression than the corresponding vector without PAS.

For eukaryotic proteins, a large number of proteins were not expressed in both vector systems (46.9%).

Like eukaryotic proteins, prokaryotic proteins had 33% higher expression rates when using the PAS vector system than conventional vectors. Specifically, Hp0096, Hp0331, Hp0572, Hp0844, Hp1069-full, Hp1069-protease and Hp1355 proteins in the PAS-N-His-Tag vector; Hp0331, Hp0572, Hp0844, Hp1069-ATP and Hp1069-full proteins for the PAS-C-His-Tag vector; Hp0332, Hp1069-full, Hp1069-protease, Hp1178, Hp1355 and Hp1496 proteins in the PAS-Nus-Tag vector; In the case of the PAS-GST-Tag vector, the Hp0175, Hp0844, Hp0925, Hp1069-ATP, Hp1069-full, Hp1178 and Hp1496 proteins showed higher expression levels in the PAS vector system than in the existing vector.

Statistical analysis of the expression results of 50 prokaryotic and eukaryotic proteins used in the performance verification showed that the expression of PAS fusion vectors was 30% higher than that of conventional vectors. It can be seen that.

Item Con> PAS Con = PAS Con <PAS Con X PAS Prokaryotes 20% 12% 33% 35% Eukaryotes 9.7% 16% 27.4% 46.9% 50 species in total 12.2% 14.8% 30% 43%

^{* In} Table 2, Con> PAS: when the expression vector of the conventional vector is more, Con = PAS: when the expression amount of the conventional vector and the PAS vector is the same, Con <PAS: when the expression of the PAS vector is more, Con X PAS : When not expressed in both conventional vector and PAS vector.

Figure 3 is a 12% acrylamide gel image showing the amount of protein induced expression of the PAS fusion vector and the conventional vector is more protein amount after the induction of PAS fusion vector expression of lane 5 than the amount of protein after the conventional vector expression of lane 3 You can see that. This shows that the PAS fusion vector can express the desired protein in higher yield than the existing vector. The fusion protein size of the PAS vector after expression induction in the gel photograph was observed at a position higher than that of the protein after induction of expression of the existing vector by adding the size of the PAS factor of 8.4 kDa (FIG. 3).

Solubility experiments were carried out by collecting proteins expressing cells in both systems, a PAS fusion vector and a conventional vector system. The cells collected after cell culture were suspended with 20 mM Tris-HCl (pH 7.5) containing 50 mM KCl, 1 mM EDTA, and 1 mM PMSF, then pulverized ultrasonically on ice and centrifuged (15,000 rpm, 1 hour). , 4 ° C.) was used to separate the supernatant from the cell residue. The solubility of the protein contained in the separated cell residue and the supernatant was analyzed by 12% acrylamide gel electrophoresis.

Since all of the proteins used in the experiments were very low solubility human and bacterial proteins, most of the proteins in the PAS fusion vector system and the existing vector system were undissolved. However, as shown in FIG. 4, in the case of some proteins, the solubility of the protein was increased when using the PAS vector system than when using the conventional vector. On the other hand, when the conventional vector was used, the solubility of the protein did not increase.

As described in detail above, the present invention provides a composition for enhancing solubility of a recombinant protein or peptide. The present invention also provides a method of increasing the solubility of a recombinant protein or peptide. According to the present invention, it is possible to obtain a recombinant protein or peptide which has not been efficiently obtained through a cloning process due to its solubility in the conventional expression system.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (10)

delete delete delete delete delete A method of increasing the solubility of a recombinant protein or peptide comprising the following steps: (a) a host with a vector comprising (i) a promoter, (ii) Plasmid Acromobacter Secretion (PAS) factor-coding nucleotide sequence operably linked to said promoter, and (iii) said recombinant protein or peptide-coding nucleotide sequence. Transforming the cell, wherein the PAS factor and recombinant protein or peptide are expressed in the form of a fusion protein; (b) culturing the transformed host cell and expressing the recombinant protein or peptide; (c) crushing the host cell to obtain a cell lysate; And (d) obtaining the recombinant protein or peptide from the cell lysate, wherein the obtained recombinant protein or peptide is in a fused form with PAS factor, and the recombinant protein or peptide in the form of the fusion protein is Solubility is increased compared to recombinant proteins or peptides that are not bound. 7. The method of claim 6, wherein said recombinant protein or peptide is Helicobacter pylori-derived Hp 0175 or human-derived ubiquitin binding enzyme (Ubiquitin-conjugation enzyme) Ku007066. 7. The glutathione S-transferase (GST), maltose binding protein (MBP), FLAG, 6x His (hexahistidine) at positions adjacent to the 5'- or 3'- terminus of the PAS factor-encoding nucleotide sequence. , Nucleotide sequence encoding NusA (N utilization substance A) or Trx (Thioredoxin). The method of claim 6, wherein the vector has a genetic map of any one of FIGS. 1A to 1E. 7. The method of claim 6, wherein the 5'-terminus of the recombinant protein or peptide-coding nucleotide sequence further comprises a Tobacco Etch Virus protease (TEV) recognition site-coding nucleotide sequence.

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