# Patent

## Title
Method for preparing soluble and active recombinant proteins usins PDI as a fusion partner

## Abstract

The present invention relates to a method for producing a recombinant protein capable of increasing expression rate of a target protein and also improving solubility and folding of the expressed target protein using a modified protein disulfide isomerase (PDI) as a fusion partner, and an expression vector containing the modified PDI gene as a fusion partner. The method for preparing a recombinant protein using a modified PDI as a fusion partner according to the present invention may solve the problems concerning a low yield and solubility and folding that conventional fusion partners have, and be widely used for protein drug and industrial protein production.

## Background
TECHNICAL FIELD
The present invention relates to a method for producing a recombinant protein, which has improved solubility and folding, as well as a high expression rate, employing protein disulfide isomerase (PDI) as a fusion partner, and an expression vector containing a PDI gene as a fusion partner.
More particularly, the present invention relates to a method for preparing a recombinant protein capable of increasing an expression rate of a desired target protein and also improving solubility and folding of the expressed target protein employing a modified PDI as a fusion partner, wherein the modified PDI is obtained by removing a ribosome binding site from a normal PDI by means of a genetic modification and adding a sequence lysine-isoleucine-glutamic acid-glutamic acid-glycine-lysine (KIEEGK (SEQ ID NO: 37), referred to as “M6”) to an amino terminus of the normal PDI, the ribosome binding site being present in common in PDI sequences derived from many species, and the target protein is produced by expressing the PDI and the target protein in a form of fusion protein in which the PDI and target protein are bound through a peptide bond, and an expression vector containing a modified PDI gene as a fusion partner.
BACKGROUND ART
Bioindustries, in which physiologically active proteins such as insulins, growth hormones, interferons, enzymes, etc. are produced in microorganisms, have grown rapidly with development of genetic recombination technologies. In recent years, high-speed/high-efficiency production of proteins has taken a very important location in various fields of structural and functional genomics, possession of target proteins for screening new drugs in various post-genome studies, etc. Until now, microorganisms (Escherichia coli, yeast), mammalian cells, etc. have been used for protein production, but protein production systems in organisms, which are the most suitable for the high-speed/high-efficiency protein production which is the heart in the studies of the functional genomics, has been known as an E-coli system which grows rapidly and is the most studied area in microbiological and physiological fields.
The protein production systems using E-coli have an excellent economic efficiency in view of the cost and accommodations, but they have one major problem that a majority of eukaryotic proteins are produced in a form of inclusion bodies, which are precipitates in cells, other than active forms since the proteins are not exactly folded into the active forms when the eukaryotic proteins are produced in cytoplasm of the prokaryotic E-coli. In order to obtain the active proteins from the inclusion bodies, the inclusion bodies should be solubilized in a high concentration of guanidine-HCl, and then refolded into an active form using methods such as dilution, etc. It has been known that large amounts of time and expense are required for finding effective refolding conditions since the refolding mechanism has not been found in full and refolding conditions are different in every protein. Highly expensive apparatuses are required for mass-production of a desired protein due to a low refolding yield of the protein, and it is difficult or impossible to refold a majority of high molecular weight proteins, which is an obstacle to industrial applications of the proteins. The inclusion body is formed since intermolecular aggregation of protein-folding intermediates appears during the folding process even if the active proteins are in the most stable form in a thermodynamic aspect [Mitraki, A. & King, J. (1989) Bio/Technology 7: 690-697]. Another reason is why disulfide bonds in the protein should be suitably formed so that the proteins can be biologically active, but the disulfide bonds in the proteins are not suitably formed in E-coli cytoplasm due to its reducing condition when the proteins are expressed in the E-coli cytoplasm.
As described above, the method, in which the genetically recombinant protein is produced in an active form, will be successfully carried out when the folding and disulfide bonding procedures are satisfied at the same time, and therefore it is difficult to produce a desired protein in the most cases. Also, it has been known that high molecular weight antibody proteins, tissue-type plasminogen activators, factor VIII, etc. are produced in forms of inclusion bodies in an E-coli system, and it is very difficult to obtain the proteins in active forms through the refolding process. In order to solve the above problems caused when a recombinant protein is produced in a form of inclusion body, it is important to express the recombinant protein in E-coli in a soluble form.
Up to now, there have been methods for expressing a recombinant protein in a soluble form: (i) the first one is a method where a recombinant protein is designed to secrete into E-coli periplasm to obtain a soluble form of the protein [Stader, J. A. & Silhavy, T. J. (1970) Methods Enzymol. 165: 166-187], but the method has a low industrial efficiency due to a low expression rate of the protein. (ii) The second one is a method where a soluble form of a recombinant protein is obtained by co-expressing a recombinant protein gene and chaperone genes such as GroEL, Dna K or the like which is involved in the protein folding [Goloubinoff, P. et al. (1989) Nature 337: 44-47], but the method is not general in preventing the formation of inclusion bodies since the method is applicable to specific proteins. (iii) The third one is a method wherein a soluble protein is obtained by selecting a protein, expressed in a soluble form in E-coli, as a fusion partner and fusing the desired recombinant protein with a carboxyl terminus of the fusion partner. Until now, the various proteins have been known as the fusion partners, including maltose-binding protein [Kapust, R. B. & Waugh, D. S. (1999) Protein Sci. 8: 1668-1674], NusA [Davis, G. D. et al. (1999) Biotechnol. Bioeng. 65: 382-388], glutathione-S-transferase [Smith, D. B. & Johnson, K. S. (1988) Gene 67: 31-40], thioredoxin [Lavallie, E. R. et al. (1993) Bio/Technology 11: 187-193], Protein-A [Nilsson, B. et al. (1985) Nucleic Acid Res. 13: 1151-1162], an amino terminal domain of transcription initiation factor IF2 [Sorensen, H. P. et al. (2003) Protein Expr. Purif. 32: 252-259], lysil-tRNA synthetase [Choi, Sung-il & Seong, Beak-Lin (1999) Korea Patent No. 10-203919], etc. However, the fusion partners have problems in view of their applications since they are merely expected to improve protein solubility but not to satisfy the protein folding and disulfide bonding at the same time.
Protein disulfide isomerase (PDI), which is an enzyme for catalyzing a thiol:disulfide bond exchange reaction, is found at a high concentration in endoplasmic reticulum in cells. It has been known that, amongst about 20 protein factors known as protein folding regulators in the cells up to the present date, proteins having a catalytic activity, such as PDI, are by no means common [Rothman, J. E. (1989) Cell 59: 591-601]. The PDI facilitates the exact formation of disulfide bonds in proteins by means of the thiol:disulfide bond exchange reaction, and serves as a chaperone when a high concentration of the PDI is present in the cells [Puig, A. & Gilbert, H. F. (1994) J. Biol. Chem. 269: 7764-7771].
Therefore, there have been attempts to produce active proteins by using the PDI. It was reported that PDI from thermophilic fungi is fused with an amino terminus of a target protein to secret a fusion protein from Bacillus brevis [Kajino, T. et al. (2000) Appl. Environ. Microbiol. 66: 638-642], or that dsbC, which is a kind of the PDI, is co-expressed with a target protein in an oxidizing cytoplasm of mutant E-coli [Bessette, P. H. et al. (1999) Proc. Natl. Acad. Sci. USA, 96: 13703-13708], etc. However, it was revealed that the target protein is produced in a very low yield since it is degraded by proteases secreted by the Bacillus strain when the target protein is secreted from the Bacillus strain. And, it was also revealed that the target protein has a very low expression rate when it is co-expressed with the dsbC in the cytoplasm.
PDI has a problem that active PDI is expressed at a low level since inactive PDI fragments are produced at the same time due to the presence of ribosome binding sites in the PDI protein. The inventors have found the fact that intact PDI protein other than split PDI protein is stably produced in a soluble form in the cytoplasm if the ribosome binding sites in the PDI protein are removed by means of a genetic modification, and they have designed a novel form of a fusion protein system that may satisfy all effects, such as high expression rate, improved solubility, protein folding, disulfide bonding, etc., by using a genetically engineered PDI as a fusion partner, the genetically engineered PDI being obtained by adding an M6 sequence (KIEEGK (SEQ ID NO: 37)) to an amino terminus of the PDI protein. All recombinant proteins, obtained using a fusion protein system with the genetically engineered PDI protein, were stably produced at a high expression rate and with a high solubility, and exhibited the same or more activity than a wild type PDI protein when the recombinant proteins were degraded through enzymatic cleavage.
DISCLOSURE OF INVENTION
Accordingly, the present invention is designed to solve the problems of the prior art, and therefore it is an object of the present invention to provide a novel method capable of stably producing the proteins at a high expression rate, as well as overcoming problems concerning a low solubility of expressed proteins and a low production of active proteins which were the most problematic when the PDI proteins are produced in E-col by satisfying a high expression rate of a target protein, improved production of soluble protein, and protein folding into an active form using PDI as a fusion partner.
In order to accomplish the above object, the present invention provides a method for stably preparing a target protein in a soluble and active form in an E-coli strain and simultaneously producing the target protein at a high expression rate using the PDI.
More particularly, the present invention provides a method for producing a fusion protein using a modified PDI protein and a polypeptide as a fusion partner and easily purifying the fusion protein through enzymatic cleavage, wherein the modified PDI protein is obtained by removing a ribosome binding site from a rat PDI gene by means of a genetic modification and adding a M6 sequence (KIEEGK (SEQ ID NO: 37)) to an amino terminus of the rat PDI gene, the polypeptide has an enzymatic cleavage site to a carboxyl terminus thereof, and the fusion protein is obtained by connecting the fusion partner with a target protein through the peptide bonds.
DNA sequences of the genetically modified rat PDI gene were designed according to the present invention and listed as follows. An underlined region is a modified gene sequence for the purpose of removing a ribosome binding site, and a darkened region is a M6 sequence added to enhance an expression rate.








   1
AAAATCGAAG AAGGTAAAGA CGCTCTGGAG GAGGAGGACA


 



ACGTCCTGGT GCTGAAGAAG


 


  61
AGCAACTTCG CAGAGCCGGC GGCGCACAAC TACCTGCTGG


 



TGGAGTTCTA TGCCCCATGG


 


 121
TGTGGCCACT GCAAAGCACT GGCCCCAGAG TATGCCAAAG


 



CTGCTGCAAA ACTGAAGGCA


 


 181
GAAGGCTCTG AGATCCGACT AGCAAAGGTG GACGCCACAG


 



AAGAGTCTGA CCTGGCCCAG


 


 241
CAGTATGGTG TCCGTGGCTA CCCCACAATC AAGTTCTTCA


 



AGAATGGAGA CACAGCCTCC


 


 301
CCAAAGGAAT ATACAGCTGG CAGGGAAGCT GACGACATTG


 



TGAACTGGCT GAAGAAACGC


 


 361
ACAGGCCCAG CAGCCACAAC CCTGTCTGAC ACTGCAGCTG


 



CAGAGTCCTT GGTGGACTCA


 


 421
AGCGAAGTGA CGGTCATCGG CTTCTTCAAG GACGCAGGGT


 



CAGACTCCGC CAAGCAGTTC


 


 481
TTGCTGGCAG CAGAGGCTGT TGATGACATA CCTTTTGGAA


 



TCACTTCCAA TAGCGATGTG


 


 541
TTTTCCAAGT ACCAGCTGGA CAAGGATGGG GTGGTCCTCT


 



TTAAGAAGTT TGATGAAGGC


 


 601
CGCAACAATT TTGAAGGTGA GATCACCAAG GAGAAGCTAT


 



TAGACTTCAT CAAGCACAAC


 


 661
CAGCTGCCTT TGGTCATCGA GTTCACTGAA CAGACAGCTC


 



CAAAGATTTT CGGAGGTGAA


 


 721
ATCAAGACAC ATATTCTGCT GTTCCTGCCC AAGAGTGTGT


 



CTGACTACGA TGGCAAATTG


 


 781
AGCAACTTTA AGAAAGCGGC CGAGGGCTTT AAGGGCAAGA


 



TCCTGTTCAT CTTCATCGAT


 


 841
AGTGACCACA CTGACAACCA GCGCATACTT GAGTTCTTTG


 



GCCTGAAGAA GGAGGAATGT


 


 901
CCAGCTGTGC GGCTTATTAC CCTTGATGAA GATATGACCA


 



AGTACAAACC GGAGTCAGAC


 


 961
GAGCTGACAG CTGAGAAGAT CACACAATTT TGCCACCACT


 



TCCTGGAGGG CAAGATCAAG


 


1021
CCCCACCTGA TGAGCCAGGA ACTGCCTGAA GACTGGGACA


 



AGCAGCCAGT GAAAGTGCTA


 


1081
GTTGGGAAAA ACTTTGAGGA GGTTGCTTTT GATGAGAAAA


 



AGAACGTGTT TGTTGAATTC


 


1141
TATGCTCCCT GGTGTGGTCA CTGCAAGCAG CTAGCCCCGA


 



TTTGGGATAA ACTGGGAGAG


 


1201
ACATACAAAG ACCATGAGAA TATCGTCATC GCTAAGATGG


 



ACTCAACAGC CAATGAGGTG


 


1261
GAAGCTGTGA AGGTGCACAG CTTTCCCACA CTCAAGTTCT


 



TCCCAGCAAG TGCAGACAGA


 


1321
ACGGTCATTG ATTACAACGG TGAGCGGACA CTAGATGGTT


 



TTAAGAAATT CTTGGAGAGC


 


1381
GGTGGCCAGG ATGGAGCGGG GGACAATGAC GACCTCGACC


 



TAGAAGAAGC TTTAGAGCCA


 


1441
GATATGGAAG AAGACGACGA TCAGAAAGCC GTGAAGGATG


 



AACTG






In addition to the rat PDI used in the present invention, many PDI gene sequences from human, cattle, fowl, orangutan, chinese hamster, rabbit, mouse, venomous snake, frog, drosophila, Fasciola hepatica, cattle parasite, hookworm, Caenorhabditis elegans, Brugia malayi, etc. have a ribosome binding site inside the PDI itself.
These sequences have in common a sequence AGGAGGAG ATG (SEQ ID NO: 42) (an underlined G is G or A) and form a ribosome binding site [Lewin, B., Genes VII, Oxford University Press, pp. 147-148]. Accordingly, a genetically modified PDI gene, in which the ribosome binding site is removed through the genetic modification, may be prepared from all the sequences in the same manner as in the rat PDI gene, and its expression may be stably induced at a high expression rate by adding the M6 sequence to an amino terminus of the modified PDI gene.
The present invention is characterized in that stability, expression rate, solubility and folding of a desired target protein is improved, and the target protein is produced in a form of a fusion protein in which an enzymatic cleavage site is inserted between the genetically modified PDI and the target protein, and then easily purified through the enzymatic cleavage. Also, a histidine tag containing 6-10 histidine residues may be added to an amino terminus or a carboxyl terminus of the genetically modified PDI, or a carboxyl terminus of the target protein for the purpose of the easy purification. In this case, the fusion protein may be easily purified using Ni-chelating affinity column chromatography. In another method used for the easy purification, a peptide sequence containing 6-10 aspartic acid and glutamic acid residues, namely DEDDDE (SEQ ID NO: 38), DEDEDE (SEQ ID NO: 39) or DEDEDEDEDE (SEQ ID NO: 40), may be added to a carboxyl terminus of the modified PDI.
A transformant may be prepared by introducing an expression vector, which produces the fusion protein, into a suitable host cell, for example E-coli strains BL21(DE3), HMS174(DE3), Rossetta(DE3), etc., and cultivated under suitable conditions to stably produce a soluble and active fusion protein containing a target protein at a high expression rate. The cultivated cells are subject to lysozyme digestion, freezing and thawing, sonication, or French press, etc., and then an aqueous solution containing the fusion protein is obtained using centrifugation, filtration, etc. The fusion protein may be easily isolated with conventional purification methods such as affinity chromatography, ion exchange chromatography, gel filtration, etc. The isolated fusion protein may be treated with a suitable amount of enterokinase or TEV protease, etc to obtain a target protein only.



BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of preferred embodiments of the present invention will be more fully described in the following detailed description, taken accompanying drawings. In the drawings:
FIG. 1 is a diagram showing an outline of constructing a plasmid vector pSSB-PDI1.
FIG. 2 a diagram showing outlines of constructing plasmid vectors pSSB-PDI1-tPA, pSSB-PDI1-EKL, pSSB-PDI1-PI and pSSB-PDI1-Bat.
FIG. 3 a diagram showing outlines of constructing plasmid vectors pSSB-PDI1-BMP2, pSSB-PDI1-Ang, pSSB-PDI1-TEV, pSSB-PDI1-IL2, pSSB-PDI1-GCSF and pSSB-PDI1-TNF.
FIG. 4 is an SDS-PAGE diagram showing that all tissue plasminogen activator (tPA) protein is found in an inclusion body (insoluble) only the tPA protein is expressed solely (Left), but a majority of the tPA protein is produced in a soluble form when it is expressed as a fusion protein with a genetically modified M6PDI (Right).
FIG. 5 is an SDS-PAGE diagram showing the total expression level and soluble and insoluble expression levels of the genetically modified M6PDI-EKL fusion protein.
FIG. 6 is an SDS-PAGE diagram after the genetically modified M6PDI-EKL (Left) and the wild type M6PDI-EKL (Right) are expressed and purified using Ni-chelating affinity column chromatography. It was shown that the wild type PDI is co-expressed with small and split PDI-EKL fragments if the wild type PDI is used as a fusion partner.
FIG. 7 is an SDS-PAGE diagram showing the total expression levels and soluble and insoluble expression levels of a solely expressed proinsulin (Left) and an M6PDI-proinsulin fusion protein (Right). It is shown that the proinsulin is expressed in a form of an inclusion body when it is expressed solely.
FIG. 8 is an SDS-PAGE diagram showing the total expression level and soluble and insoluble expression levels of an M6PDI-Batroxobin fusion protein.
FIG. 9 is an SDS-PAGE diagram showing the total expression level and soluble and insoluble expression levels of an M6PDI-BMP2 fusion protein.
FIG. 10 is an SDS-PAGE diagram showing the total expression levels and soluble and insoluble expression levels of a solely expressed angiogenin and an M6PDI-Angiogenin fusion protein. It is shown that the angiogenin is expressed in a from of an inclusion body when it is expressed solely.
FIG. 11 is an SDS-PAGE diagram showing the total expression level and soluble and insoluble expression levels of an M6PDI-TEV protease fusion protein. It is shown that the M6PDI and the TEV protease are produced in a cleaved form.
FIG. 12 is an SDS-PAGE diagram showing the total expression levels and soluble and insoluble expression levels of an M6PDI-IL2 fusion protein.
FIG. 13 is an SDS-PAGE diagram showing the total expression levels and soluble and insoluble expression levels of an M6PDI-GCSF fusion protein.
FIG. 14 is an SDS-PAGE diagram showing the total expression levels and soluble and insoluble expression levels of an M6PDI-TNF fusion protein.
FIG. 15 is a diagram showing comparison between activities of a commercially available wild type tPA and the M6PDI-tPA of the present invention. It might be seen that the M6PDI-tPA of the present invention has a similar activity to the wild type tPA even though it is expressed in a fused form.
FIG. 16 is a diagram showing that the fusion protein is cleaved into an active enterokinase light chain (EKL) protein with the passage of time.
FIG. 17 is a bar graph showing comparison between activities of recombinant angiogenin (rbAng) produced according to the present invention and wild type angiogenin (nbAng) purified from cow mil. It might be seen that the recombinant angiogenin has a better activity than that of the wild type angiogenin.



BEST MODES FOR CARRYING OUT THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the description proposed herein is just a preferable example for the purpose of illustrations only, not intended to limit the scope of the invention, so it should be understood that other equivalents and modifications could be made thereto without departing from the spirit and scope of the invention.
Embodiment 1
Construction of pSSB-PDI1 Plasmid Vector
A molecular genetic technique used in the present invention is based on a literature [Ausubel, F. M. et al. (Ed.), J. Wiley Sons, Curr. Protocols in Molecular Biology, 1997]. Primers used for a polymerase chain reaction (PCR) were ordered and synthesized at Bioneer Corp., rTaq polymerase was commercially available from TaKaRa, and PCR was carried out according to a standard condition presented by a TaKaRa's manual protocol.
A PDI gene was cloned by carrying out PCR using cDNA of a rat PDI gene as a template. A fusion protein was designed so that a sequence of 6 amino acid residues (M6) existing in an amino-terminal domain of maltose-binding protein was added to an amino terminus of a PDI protein so as to enhance an expression rate of a PDI protein, and a peptide sequence containing 6 aspartic acid and glutamic acid residues and a linker amino acid residue was added to a carboxyl terminus of the PDI protein for the purpose of its easy purification. At this time, oligonucleotides set forth in SEQ ID NOs: 1 and 2 were used as the primers.
Since the rat PDI gene has a ribosome binding site in the middle thereof, the rat PDI gene was co-expressed with a small-sized PDI fragment, which does not have an activity as the fusion partner. Therefore, in order to remove the ribosome binding site located in the middle of the PDI gene, bases at 911th, 914th and 920th nucleotides of the PDI gene were modified using a PCR method using primers set forth in SEQ ID NOs: 3 and 4 (a genetically modified PDI gene: SEQ ID NO: 25 and its protein: SEQ ID NO: 26). At first, the primers set forth in SEQ ID NOs: 1 and 4 were dissolved in a TE (pH 8.0) solution to a density of 10 picomole/μl, respectively, and then PCR was carried out using the primers to amplify a DNA fragment containing the upstream part of the PDI gene. Subsequently, PCR was carried out using equivalent amounts of the primers set forth in SEQ ID NOs: 2 and 3 to amplify a DNA fragment containing the downstream part of the PDI gene. Exact sizes of the DNA fragments were determined in DNA agarose gel (1×TAE, 1% agarose), and DNA bands in the agarose gel were cut and purified with a gel extraction kit (Intron), respectively. The two DNA fragments were mixed at an equivalent amount to obtain a template, and then PCR was carried out using the template and primers set forth in SEQ ID NOs: 1 and 2 to obtain an amplified DNA fragment corresponding to a final M6PDI gene (see FIG. 1).
1 μg of the amplified DNA fragment was dissolved in 50 μl TE (pH 8.0) solution and mixed with 2 units of NdeI (NEB) and 2 units of KpnI (NEB), and then the resultant mixture was reacted at 37° C. for 16 hours to obtain a DNA fragment having an NdeI restriction enzyme recognition site at its 5′-terminus and a KpnI restriction enzyme recognition site at its 3′-terminus. Only the DNA fragment was purified using a DNA purification kit (intron), and a linear pGEMEX-1 plasmid (Promega) was prepared by treating a circular pGEMEX-1 plasmid with restriction enzymes NdeI and KpnI in the same manner as in the above. Subsequently, 20 ng of the DNA fragment and 20 ng of the linear pGEMEX-1 plasmid were mixed in 10 μl TE (pH 8.0) solution, and then the resultant mixture was added 1 unit of T4 DNA ligase (NEB) and reacted at 16° C. for 16 hours to ligate them into one plasmid. The plasmid prepared thus was named pSSB-PDI1 (see FIG. 1).
Embodiment 2
Construction of Expression Plasmid for Production of Tissue-type Plasminogen Activator in Form of Fusion Protein
A human tissue-type plasminogen activator (hereinafter, referred to as “tPA”) gene was used as a template, and a genetically modified tPA was also used as a template to enhance an expression rate of a target protein considering codon usage.
In order to produce the target protein in a form of fusion protein and remove a fusion partner from the fusion protein, a tPA gene containing a DNA sequence at its 5′-terminus was amplified with a PCR method using primers set forth in SEQ ID NOs: 5 and 6, the DNA sequence encoding an amino acid sequence recognized by TEV enzyme.
1 μg of the amplified DNA fragment was dissolved in 50 μl TE (pH 8.0) solution, and mixed with 2 units of KpnI (NEB), and then the resultant mixture was reacted at 37° C. for 6 hours, and only a DNA fragment was purified with a DNA purification kit (Intron). Also, the purified DNA was dissolved in 50 μl TE (pH 8.0) solution, and mixed with 2 units of SalI (NEB), and then the resultant mixture was reacted at 37° C. for 16 hours to obtain a DNA fragment having a KpnI restriction enzyme recognition site at its 5′-terminus and a SalI restriction enzyme recognition site at its 3′-terminus. The DNA fragment was inserted to the plasmid pSSB-PDI1 which was treated with restriction enzymes KpnI and SalI in the same manner as in the above. The expression plasmid prepared thus was named pSSB-PDI1-tPA (see FIG. 2).
Embodiment 3
Construction of Expression Plasmid for Production of EKL in Form of Fusion Protein
EKL is a light chain domain which is an active region of the protease, bovine enterokinase, and only the light chain of the bovine enterokinase was used as a template to clone an EKL gene.
In order to produce the target protein in a form of fusion protein and remove a fusion partner from the fusion protein, an EKL gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 7 and 8, the EKL gene containing a DNA sequence, which encodes an amino acid sequence (DDDDK (SEQ ID NO: 43)) recognized by EKL enzyme, at its amino-terminal domain, and a DNA sequence containing 6 histidine residues at its carboxyl-terminal domain for the purpose of improving its purification.
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-EKL (see FIG. 2).
Embodiment 4
Construction of Expression Plasmid for Production of Proinsulin in Form of Fusion Protein
Human proinsulin cDNA was used as a template to clone a proinsulin gene.
For the purpose of isolation from a fusion partner and production of insulin, a proinsulin gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 9 and 10, the proinsulin gene containing a DNA sequence encoding 8 histidine residues and 2 arginine residues (RR).
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-PI (see FIG. 2).
Embodiment 5
Construction of Expression Plasmid for Production of Batroxobin in Form of Fusion Protein
Batroxobin cDNA extracted from a snake Bothrops atrox moojeni was used as a template to clone a batroxobin gene [see Itoh, N., Tanaka, N., Mihashi, S. and Yamashina, I. (1987) J. Biol. Chem. 262, 3125-3132].
For the purpose of isolation from a fusion partner, a batroxobin gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 11 and 12, the batroxobin gene containing a DNA sequence encoding a sequence of amino acid residues (ENLYFQ (SEQ ID NO: 44)) recognized by TEV protease.
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-Bat (see FIG. 2).
Embodiment 6
Construction of Expression Plasmid for Production of Bone Morphogenetic Protein-2 (BMP-2) in Form of Fusion Protein
Human bone morphogenetic protein-2 (hBMP-2) cDNA was used as a template to clone a BMP-2 gene.
For the purpose of isolation from a fusion partner, a BMP-2 gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 13 and 14, the BMP-2 gene containing a DNA sequence encoding a sequence of amino acid residues (ENLYFQ (SEQ ID NO: 44)) recognized by TEV protease.
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-BMP2 (see FIG. 3).
Embodiment 7
Construction of Expression Plasmid for Production of Angiogenin in Form of Fusion Protein
Bovine-derived angiogenin cDNA was used as a template to clone an angiogenin gene.
For the purpose of isolation from a fusion partner, an angiogenin gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 15 and 16, the angiogenin gene containing a DNA sequence encoding a sequence of amino acid residues (ENLYFQ (SEQ ID NO: 44)) recognized by TEV protease.
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-Ang (see FIG. 3).
Embodiment 8
Construction of Expression Plasmid for Production of Tobacco Etch Virus (TEV) Protease in Form of Fusion Protein
27 kDa catalytic domain cDNA of NIa (Nuclear Inclusion a), extracted from tobacco etch virus (TEV), was used as a template to clone a TEV protease gene.
For the purpose of isolation from a fusion partner, a TEV protease gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 17 and 18, the TEV protease gene containing a DNA sequence encoding a sequence of amino acid residues (ENLYFQ (SEQ ID NO: 44)) recognized by TEV protease.
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-TEV (see FIG. 3).
Embodiment 9
Construction of Expression Plasmid for Production of Interleukin-2 (IL2) in Form of Fusion Protein
Human interleukin-2 cDNA was used as a template to clone an IL2 gene.
For the purpose of isolation from a fusion partner,an IL2 gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 19 and 20, the IL2 gene containing a DNA sequence encoding a sequence of amino acid residues (ENLYFQ (SEQ ID NO: 44)) recognized by TEV protease.
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-IL2 (see FIG. 3).
Embodiment 10
Construction of Expression Plasmid for Production of Granulocyte Colony Stimulating Factor (GCSF) in Form of Fusion Protein
A PCR method was carried out using human GCSF cDNA as a template to obtain a granulocyte colony stimulating factor (GCSF) gene.
In order to express a target protein and isolate the target protein from a fusion partner, a GCSF gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 21 and 22, the GCSF gene containing a DNA sequence encoding a sequence of amino acid residues (ENLYFQ (SEQ ID NO: 44)) recognized by TEV protease.
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-GCSF (see FIG. 3).
Embodiment 11
Construction of Expression Plasmid for Production of Tumor Necrosis Factor-Alpha (TNF-α) in Form of Fusion Protein
Human TNF-α (human Tumor Necrosis Factor-α) cDNA was used as a template to clone a tumor necrosis factor-alpha gene.
In order to express a target protein and isolate the target protein from a fusion partner, a TNF-α gene was amplified with a PCR method using primers set forth in SEQ ID NOs: 23 and 24, the TNF-α gene containing a DNA sequence encoding a sequence of amino acid residues (ENLYFQ (SEQ ID NO: 44)) recognized by TEV protease.
The amplified DNA fragment was inserted between sites of the plasmid pSSB-PDI1 recognized by restriction enzymes KpnI and SalI in the same manner as in Embodiment 2. The plasmid vector prepared thus was named pSSB-PDI1-TNF (see FIG. 3).
Embodiment 12
Preparation of E-coli Transformant
A typical production strain E-coli BL21(DE3), HMS174(DE3) or Rossetta(DE3) was transformed respectively with the expression plasmid pSSB-PDI1-(tPA, EKL, PI, Bat, BMP2, Ang, TEV, IL2, GCSF, and TNF), prepared in Embodiments 2 to 11, using a method proposed by Hanahan, and ampicillin-resistant colonies were selected, respectively [Hanahan, D. (1985) DNA Cloning vol. 1 (Ed. D. M. Glover) 109-135, IRS press].
The strain E-coli Rosetta(DE3) transformed with the expression vector pSSB-PDI1-tPA was selected and deposited in an international depository authority, the Korean Culture Center of Microorganisms (KCCM, #361-221, Yurim Building, Hongje-1-dong, Seodaemun-gu, Seoul, Republic of Korea) on Jan. 27, 2005 under an accession number of KCCM-10646P according to the Budapest Convention.
Embodiment 13
Cell Culture and Production of Target Protein
Each of the E-coli transformants transformed respectively with the recombinant expression vectors pSSB-PDI1-(tPA, EKL, PI, Bat, TEV, ANG, IL2, BMP-2, GCSF and TNF), prepared in Embodiments 2 to Embodiment 11, was inoculated and cultured in a liquid medium (tryptone 10 g/l, yeast extract 10 g/l, sodium chloride 5 g/l) containing ampicillin (50 ̃100 μg/ml) or ampicillin and chloramphenicol (38 ̃50 μg/ml).
The recombinant E-coli strains were cultured in the liquid medium, and then in a solid medium containing the same components as in the solid medium, and the resultant colonies were cultivated for 12 hours in 1 μl of a liquid medium containing ampicillin (50 ̃100 μg/ml) or ampicillin and chloramphenicol (38 ̃50 μg/ml), and then the colony culture medium was suspended in 15% glycerol solution, which was stored at −70° C. for future use. The recombinant E-coli strains stored at −70° C. were spread on the same solid medium as in the above and cultivated at 37° C. for 16 ̃18 hours to allow colonies to grow on the solid medium, and then the grown colonies were inoculated again in 20 ml liquid medium and cultivated at 37° C. while stirring at a rotary speed of 200 rpm. 16 ̃17 hours after their cultivation, the resultant liquid mediums were inoculated in 400 ml of a liquid medium to a density of 5%, and cultivated at 37° C. while stirring at a rotary speed of 200 rpm, pH 7. When the recombinant E-coli strains were grown to an optimal density of 0.4 ̃0.6 at 600 nm, isopropyl-3-D-thiogalactopyranoside (IPTG, to a final density of 0.5 ̃1 mM) was added to the culture solutions, respectively, and then suspended at 20 ̃25° C. for 4 hours while stirring at a rotary speed of 200 rpm to induce expression of fusion proteins. The resultant culture solutions were centrifuged for 10 minutes at a rotary speed of 6,000 rpm to obtain E-coli pellets, and the pellets were suspended in 20 ml of 50 mM TrisHCl buffer (pH 8.0) and then lysed with a sonication method. The cell lysates lysed with the sonication were centrifuged at 4° C. for 10 minutes at a rotary speed of 13,000 rpm to separate a supernatant fraction and a pellet fraction, and then amounts of fusion proteins in the supernatant fraction and the pellet fraction were determined on SDS-PAGE. As a result, a majority of the fusion proteins were accumulated in a soluble form (FIG. 4 through FIG. 14).
Embodiment 14
Purification and Activity of tPA Protein
20 ml of the lysed E-coli cell suspension, in which the M6PDI-tPA protein was expressed in a soluble form according to the method described in Embodiment 13, was centrifuged at a rotary speed of 13,000 rpm to separate a supernatant and a precipitate, and then the supernatant and the precipitate were subject to SDS-PAGE to determine whether the M6PDI-tPA protein is expressed in a soluble form (see a right part of FIG. 4). As a result, it was proven that the M6PDI-tPA protein of the present invention is excellent in that the fusion protein is expressed in a soluble form, on the contrary to the fact that all the tPA protein is in an insoluble precipitate fraction when it is expressed solely (see a left part of FIG. 4).
20 ml of the supernatant containing the M6PDI-tPA was filled in a Q-Sepharose cation exchange chromatography column, and then the M6PDI-tPA protein was isolated from the E-coli-derived proteins by allowing 50 mM TrisHCl buffer (pH 8.0) to flow through the column with a linear gradient of 0-0.5 M sodium chloride at a constant flow rate of 0.5 ml/min. TEV protease (0.7 mg/ml) with a histidine tag was added to 20 ml of the M6PDI-tPA-containing fractions as much as 10% of the total reaction solution, and reacted at 4° C. for 16 hours in 50 mM TrisHCl (pH 8.0) buffer to separate tPA protein from the fusion protein (M6PDI), and then Q-Sepharose cation exchange chromatography using isoelectric point differences between the M6PDI and the tPA was carried out in the same manner as described above to purify the tPA protein.
Activity of the purified tPA protein was estimated by measuring fibrinolytic activity [Astrup, T. & Mullertz, S. (1952) Arch. Biochem. Biophys. 40: 346-51]. 3 units of human thrombin (Hyphen BioMed) was mixed with human fibrinogen (Hyphen BioMed) dissolved in 10 ml of 50 mM TrisHCl (pH 8.0) solution to a final density of 0.7%, and then carefully poured on a plate without delay and made hard at 4° C. overnight to prepare a fibrin plate. Equivalent amounts (5 to 10 ml) of the purified tPA and a commercially available tPA (Genentech) were spotted on a surface of the plate without its spreading. An activity of the produced tPA was evaluated by comparing to that of the commercially available tPA using a method where the purified tPA and the commercially available tPA were reacted at 37° C. for 6 hours and dissolution areas of their spotted points were measured, respectively. The fibrinolytic activity was measured at an optical density of 600 nm (FIG. 15). As the result of comparing their activities, it was revealed that the tPA protein produced according to the present invention has the same activity as that of the commercially available tPA protein.
Embodiment 15
Purification and Activity of EKL Protein
The E-coli tranformant, in which the M6PDI-EKL protein prepared in the method of Embodiment 13 is accumulated in a soluble form, was harvested. 400 μml of the culture solution was centrifuged for 10 minutes at a rotary speed of 6,000 rpm to obtain an E-coli pellet, and then the E-coli pellet was suspended in 20 ml of 50 mM TrisHCl buffer (pH 8.0) and lysed with a sonication method. The cell lysate was centrifuged again to separate a supernatant and a pellet, and then the supernatant and the pellet were subject to SDS-PAGE to determine whether the M6PDI-EKL protein is expressed in a soluble form (FIG. 5).
In order to determine whether a ribosome binding site is effectively removed, a wild type PDI-EKL and a genetically modified M6PDI-EKL were expressed and purified with Ni-chelating affinity column chromatography, respectively, and then their expression results were compared to determine whether a ribosome binding site is removed (see FIG. 6).
A carboxyl-terminal domain of amino acid residues (DDDDK (SEQ ID NO: 43)) inserted between the M6PDI and the EKL was self-cleaved to separate the active EKL from the M6PDI by keeping 20 ml of the supernatant containing the M6PDI-EKL at 4° C. for 4 days (FIG. 16). The active EKL was purified with Ni-chelating affinity column chromatography using a histidine tag positioned at carboxyl terminus of the active EKL. The purification conditions are as follows: 20 ml of the supernatant was mixed with 10 ml of nickel-resin in a buffer containing 50 mM TrisHCl (pH 8.0), 500 mM sodium chloride and 10% glycerol, and stirred at 4° C. for 12 ̃16 hours to chelate the EKL with nickel, and the resultant reaction solution was washed with 100 ml, of 10 mM imidazole solution, and then the EKL protein was eluted with a linear gradient of 10-250 mM imidazole solution. 5 ml of a fraction containing the eluted EKL protein was dialyzed at 4° C. in 50 mM TrisHCl (pH 8.0) buffer to remove salts and imidazole from the solution.
An activity of the EKL purified in the present invention was compared with that of the commercially available EKL product (Invitrogen). Glycine-aspartic acid-aspartic acid-aspartic acid-aspartic acid-lysine-beta-naphtylamide (GDDDDK-β-naphtylamide (SEQ ID NO: 45), Sigma) was used as a substrate, and 0.1 ̃2 μg of the EKL protein was added to 400 μl of a buffer (pH 8.0) containing 0.5 mM substrate, 10 mM calcium chloride, 10% DMSO and 25 mM TrisHCl, and then the resultant mixture was excited at 337 nm while its being reacted at 30° C. for 1 minute, and its emission fluorescence was measured at 420 nm. As the result of measuring activity, it was shown that the EKL protein of the present invention has the same activity as that of the commercially available EKL product.










TABLE 1







Sample
ΔAU/ng









EKL from Invitrogen
0.2



EKL of the present invention
0.2










Embodiment 16
Purification and Activity of Angiogenin
The E-coli tranformant, in which the M6PDI-Ang protein prepared in the method of Embodiment 13 is accumulated in a soluble form, was harvested. 400 μml of the culture solution was centrifuged for 10 minutes at a rotary speed of 6,000 rpm to obtain an E-coli pellet, and then the E-coli pellet was suspended in 30 ml of 30 mM TrisHCl buffer (pH 8.0) containing 300 mM sodium chloride and lysed with a sonication method. The cell lysate was centrifuged for 10 minutes at a rotary speed of 13,000 rpm again to separate a supernatant and a pellet, and then the supernatant containing the M6PDI-Ang protein was purified with Ni-chelating affinity column chromatography using a histidine tag. The purification conditions are as follows: 30 ml of the supernatant was mixed with 15 ml of nickel-resin in a buffer containing 30 mM TrisHCl (pH 8.0) and 300 mM sodium chloride, and stirred at 4° C. for 12 ̃16 hours to chelate the EKL with nickel, and the resultant reaction solution was washed with 150 ml of 40 mM imidazole solution, and then the M6PDI-Ang protein was eluted with a linear gradient of 40-80 mM imidazole solution. A fraction containing the eluted M6PDI-Ang protein was dialyzed at 4° C. in 30 mM TrisHCl (pH 8.0) buffer to remove salts and imidazole from the solution.
A histidine-tagged TEV protease (0.7 mg/ml) was added to a reaction solution as much as 4% of the total reaction solution, reacted at 22° C. for 22 hours in 30 mM TrisHCl (pH 8.0) buffer to cleave a fusion partner (M6PDI) from angiogenin, and then finally purified with high performance liquid chromatography (HPLC). A column, ZORBAX SIL (C8) (Agilent), was used in the high performance liquid chromatography, the purification conditions are as follows: 85% of solution A (99.9% H2O, 0.1% TFA) and 15% of solution B (99.9% Acetonitrile, 0.1% TFA) flow through the column for 15 minutes at a constant flow rate of 0.8 ml/min, and then the solution B flow through the column for 120 minutes with a linear gradient from 15% to 29% to separate angiogenin.
An activity of the purified recombinant angiogenin was compared with that of the wild type angiogenin isolated from milk. The angiogenin activity was measured using a chick chorioallantoic membrane (CAM) model [Nguyen, M. et al. (1994) Microvasc. Res. 47: 31-40]. As the result of measuring activity, it was shown that the angiogenin prepared in the present invention has the more excellent activity than that of the wild type angiogenin (FIG. 17).
INDUSTRIAL APPLICABILITY
As described above, the method for preparing a recombinant protein according to the present invention may be useful to solve the problems caused in producing the fusion partner in an E-coli system, for example a low solubility and a low production of active proteins, and be also widely used for protein drug and industrial protein production by satisfying an improved expression rate of a target protein, an enhanced production of soluble proteins, and protein folding into an active form using a genetically modified PDI as a fusion partner.

## Claims

1. A method for preparing a recombinant protein that improves productivity, solubility and folding of a target protein, the method comprising:
(a) inserting into a vector a gene encoding protein disulfide isomerase (PDI) as a fusion partner; and
(b) ligating a gene encoding the target protein with said gene encoding PDI, wherein the amino-terminal domain of said PDI comprises a sequence of 6 amino acid residues KIEEGK (SEQ ID NO: 37) obtained from an amino-terminal domain of a maltose-binding protein, and wherein the recombinant protein comprises a sequence of 6 to 10 histidines at the amino-terminus or carboxy-terminus of the PDI protein, and a peptide sequence consisting of 6 to 10 aspartic acid and glutamic acid residues between said PDI and the target protein.

2. The method for preparing a recombinant protein according to claim 1, wherein the PDI protein is encoded by a gene sequence set forth in SEQ ID NO: 25.

3. The method for preparing a recombinant protein according to claim 1, wherein the target protein is selected from the group consisting of tissue-type plasminogen activator.

4. The method for preparing a recombinant protein according to claim 3, wherein the tissue-type plasminogen activator has the amino acid sequence of SEQ ID NO: 27.

5. The method for preparing a recombinant protein according to claim 1 further comprising a step of transforming a host cell with a recombinant expression vector containing a fused gene in which the gene encoding the target protein is ligated with the gene encoding the PDI.

6. The method for preparing a recombinant protein according to claim 5, wherein the recombinant expression vector is selected from the group consisting of pSSB-PDI1-tPA.

7. The method for preparing a recombinant protein according to claim 5, wherein the host cell is Escherichia coli.

8. The method for preparing a recombinant protein according to claim 7, wherein the host cell is a strain E-coli KCCM-10646P transformed with the expression vector pSSB-PDI1-tPA.

9. A recombinant protein prepared according to the method as defined in claim 1.

10. A nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising protein disulfide isomerase ligated to a target protein; wherein the amino-terminal domain of said PDI comprises a sequence of 6 amino acid residues KIEEGK (SEQ ID NO: 37) obtained from an amino-terminal domain of a maltose-binding protein; wherein the recombinant protein comprises a sequence of 6 to 10 histidines at the amino-terminus or carboxy-terminus of the PDI protein, and a peptide sequence consisting of 6 to 10 aspartic acid and glutamic acid residues between said PDI and the target protein.

11. A recombinant expression vector comprising the nucleic acid of claim 10.

12. A host cell transformed with the recombinant expression vector of claim 11.