PAS MUTANTS AND VECTORS CARRYING THE SAME

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to PAS (Plasmid Acromobacter Secretion) factor mutants, particularly to a PAS factor mutant, vectors carrying the same, and processes for preparing recombinant peptides and proteins using the vectors.

DESCRIPTION OF THE RELATED ART One of main reasons for performing gene cloning in the biotechnological field is to express useful genes in a microbe and produce proteins in a massive manner. Although a protein market size in the biotechnological industry could not be accurately evaluated, it is evident that protein products occupy about 60% of the total market size. In addition, the market size of proteins would be expected to sharply increase in the near future and their marketing territory will become wider because their novel uses and improvements have been continuously suggested.

For developing novel recombinant microbes to massively produce useful proteins, lots of researches have been focused on preparing recombinant host cells for stably providing foreign proteins and expression vector for secreting/expressing foreign genes in an efficient manner.

However, a variety of proteins have been reported not to express in a suitable level. Where expressed in a desirable amount, they are unlikely to be properly folded to form inclusion bodies, resulting in loss of their functions.

Vibrio septicemia is caused by infection of Vibrio vulnificus in fishes and shellfishes or seawater. Since vibrio septicemia has a short latent period and is developed at higher rate, the therapy with antibiotics is likely to be untimely. The mortality of this disease is significantly high (40-50%) and its early diagnosis and rapid treatments are therefore required. Vibrio septicemia has been repotted to be

frequently developed in countries having dietary life to eat uncooked fishes and shellfishes such as Japan and Korea, in particular, in the summer. Also, the development cases of vibrio septicemia become increased over worldwide.

Vibrio vulnificus causing vibrio septicemia is a Gram negative bacterium living in marine habitats and is well proliferated in media containing 1-3 %dls NaCI. It had been erroneously classified for a long time, and then was correctly named V. vulnificus in the year of 1979 based on the fact that it decomposes lactose unlike V. parahemolyticus. The bacterium is cloistin-resistant and ampicillin- and carbenicillin- sensitive, which distinguishes over other similar bacteria. Generally, this bacterium has a bending sausage shape and its biomass is surrounded by capsule. Vibrio vulnificus having capsular material shows resistance to antibiotic action of serum and anti-phagocytosis; therefore, it exhibits higher lethality and penetration potency to tissues. Toxins implicated in symptoms of Vibrio vulnificus infection are divided three classes: endotoxins, enzymes and other factors. Upon infection of Vibrio vulnificus into human, these toxins are highly expressed because of its survival under sudden environmental changes.

Various toxins are expressed for infection of Vibrio vulnificus into human body. Of them, PAS (Plasmid Acromobacter Secretion) factor is a small protein consisting of 76 amino acid residues. Vibrio vulnificus does not express PAS factor in natural habitats; however, upon infection into human body, it expresses excess PAS factor.

PAS factor is not expressed by in vitro cultured Vibrio vulnificus. Where Vibrio vulnificus is transformed with plasmids carrying PAS factor-encoding gene, it expresses PAS factor under overexpression conditions. The expressed PAS factor is localized in periplasmic space (see Tokugawa, K., et al., J. Biotechnol., 35:69- 76( 1994); Tokukawa, K. et al., J. Biotechnol. 37:33-37(1994)).

Based on the findings that the overexpressed PAS factor is largely observed in periplasmic space than cytoplasm, it could be recognized that most of PAS factor expressed is migrated into periplasmic space immediately after production. These

discoveries demonstrate that PAS factor is a protein not only to serve secretion of toxins produced in bacterial cells but also to serve as chaperon. In addition, it could be appreciated that PAS factor is readily folded into a stable structure and secreted with no help of leader sequences.

Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVETNION

The present inventors have made intensive researches to develop expression vectors capable of expressing recombinant peptides or proteins in much higher level. As a result, we have discovered that peptides or proteins fused to a PAS factor mutant are expressed in much higher level.

Accordingly, it is an object of this invention to provide a PAS (Plasmid Acromobacter Secretion) factor mutant.

It is another object of this invention to provide a nucleic acid molecule encoding the PAS factor mutant.

It is still another object of this invention to provide a vector carrying the nucleotide sequence encoding the PAS factor mutant.

It is further object of this invention to provide a transformant comprising the vector. It is still further object of this invention to provide a method for producing a recombinant peptide or protein.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and

drawings.

In one aspect of this invention, there is provided a PAS (Plasmid Acromobacter Secretion) factor mutant comprising the amino acid sequence of SEQ ID NO:2.

The present inventors have made intensive researches to develop expression vectors capable of expressing recombinant peptides or proteins in much higher level. As a result, we have discovered that peptides or proteins fused to a PAS factor mutant are expressed in much higher level.

The present inventors had first isolated a PAS factor as specific secretion promoting factors from Vibrio vulnificus and prepared PAS fusion vectors containing the PAS factor (see Korean Patent Appln. Pub. 2006-0006204). However, the wild type PAS factor shows no sufficient performance as expression facilitators. In these contexts, the present inventors have modified the wild type PAS factor to provide a mutant PAS factor exhibiting higher efficiency for expressing peptides or proteins.

The PAS factor mutant of this invention has a substitution in which a hydrophilic amino acid, aspartic acid positioned at amino acid 36 of the wild type PAS factor is replaced by a hydrophobic amino acid, proline. This substitution strategy has been made based on the structural analysis of PAS factor. The substitution is responsible for enhancement of binding strength of the PAS factor to cell membrane and improvement in its chaperone function, resulting in significant improvement in PAS fusion expression vectors comprising the PAS factor mutant.

In another aspect of this invention, there is provided a nucleic acid molecule comprising the nucleotide sequence encoding the PAS factor mutant.

Most preferably, the nucleic acid molecule of this invention comprises the

nucleotide sequence of SEQ ID NO:1.

The term used herein "nucleic acid molecule" is intended to encompass DNA and RNA molecules, including known analogs of natural nucleotides unless otherwise indicated (Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman and Peyman, Chemical Reviews, 90:543-584(1990)).

It would be obvious to those skilled in the art that the nucleotide sequence coding for the PAS factor mutant of this invention is not limited to the nucleotide sequence of SEQ ID NO:1.

The variations in nucleotide sequences may not cause changes in protein or peptide functions. Such a variation includes nucleic acid molecules comprising codons encoding functionally equivalent amino acids or identical amino acids {e.g., as a result of the degeneracy of genetic codes), or biologically equivalent amino acids. Furthermore, the variations in nucleotide sequences may cause changes in the

PAS factor mutant. However, such variants may provide PAS factor mutants with substantially unimpaired activity.

In still another aspect of this invention, there is provided a vector having the nucleic acid molecule comprising the nucleotide sequence encoding the PAS factor mutant.

The vector system of this invention may be constructed according to the known methods in the art as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001), which is incorporated herein by reference.

Typically, the vector may be constructed for cloning or expression. Preferably, the vector of this invention is constructed to express recombinant peptides or proteins of interest. In addition, the vector may be constructed for use in prokaryotic or eukaryotic host cells.

For example, where the vector is constructed for expression in prokaryotic cells, it generally carries a strong promoter to initiate transcription (e.g., tac

promoter, lac promoter, lacUV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site or translation initiation and a transcription/translation termination sequence. In particular, where E coli is used as a host cell, a promoter and operator in operon for tryptophan biosynthesis in E coli (Yanofsky, C, J. Bacteriol., 158:1018-1024(1984)) and a leftward promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet, 14:399- 445(1980)) may be employed as a control sequence.

Numerous conventional vectors used for prokaryotic cells are known to those of skill in the art, and the selection of an appropriate vector is a matter of choice. Conventional vector used in this invention includes plasmids {e.g., pSClOl, CoIEl, pBR322, pUC8/9, pHC79, pUC19 and pET series), phages {e.g., λgt4λB, λ-Charon, λ δzl and M13) and viruses {e.g., SV40).

For example, where the expression vector is constructed for eukaryotic host cell, inter alia, animal cell, a promoter derived the genome of mammalian cells (e.g., metallothionein promoter) or mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) may be used. The vector generally contains a polyadenylation site of the transcript. In addition, the expression vector of this invention further comprises a nucleotide sequence to conveniently purify the fusion protein expressed, which includes but not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), FLAG, 6 x His (hexahistidine), NusA and Trx. Due to the additional sequence, the fusion protein expressed can be purified with affinity chromatography in a rapid and feasible manner.

According to a preferred embodiment of this invention, the fusion protein is purified by affinity chromatography. For example, in case of using glutathione S- transferase, elution buffer containing glutathione is employed and in case of using

6X His, Ni-NTA His-binding resin is generally employed to purify the fusion protein of interest in a rapid and feasible manner.

It is preferable that the expression vector of this invention carries one or more markers which make it possible to select the transformed host, for example, genes conferring the resistance to antibiotics such as ampicillin, gentamycine, carbenicllin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

According to a preferred embodiment, the vectors of this invention further comprises protease recognition site (e.g., TEV recognition site), e.g., at downstream of sequences for purification. The fusion peptides or proteins expressed by the present vectors can be obtained in an isolated form from other sequences by protease treatment.

According to a preferred embodiment, the vector comprises an expression construct containing (i) the PAS factor mutant-encoding nucleotide sequence-(ii) a fusion partner protein-encoding nucleotide sequence-(iii) the recombinant peptide or protein-encoding nucleotide sequence. The vector of this invention is illustrated in

Rg. 3.

The peptide or protein-encoding nucleotide sequence contained in the present vector is not limited, including nucleotide sequences coding for hormones, hormone analogues, enzymes, enzyme inhibitors, signal transduction proteins or fragments thereof, antibodies or fragments thereof, single chain antibodies, binding proteins or fragments thereof, peptides, antigens, adhesive proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcription regulatory proteins, blood clotting proteins or plant defense-inducing proteins.

The peptide-encoding nucleotide sequence contained in the present vector is not limited, preferably, including nucleotide sequences coding for peptide fragments with inherent activities derived from the wild type proteins.

The vectors containing the PAS factor mutant-encoding sequence enable to express peptides or proteins of interest in much higher yield. Surprisingly,

transmembrane proteins such as ryanodine receptor and human-origiated proteins such as calcipressin that have been known not to be expressed in conventional vector systems, can be successfully expressed in the present vectors.

In further aspect of this invention, there is provided a transformant comprising the present vector.

The hosts useful in preparing the transformant are well known to those skilled in the art. For example, as prokaryotic host, E α?// JM 109, E coli BL21(DE3), E coli DH5α, E coli RRl, £ cσ// LE392, £ coli Z, E coli X 1776, £ cø// W3110, Bacillus subtilis, Bacillus thurigensis, Salmonella typhimurium, Serratia marcescens and various Pseudomonas.

As eukaryotic cell, yeast {Saccharomyce cerevisiae), insect cell and human cell (e.g., CHO (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) may be used. The transformation of a host cell can be carried out by a large number of methods known to one skilled in the art. For example, in case of using prokaryotic cells as host, CaCI ₂ method (Cohen, S.N. et al., Proc. Natl. Acac. Sd. USA, 9:2110- 2114(1973)), Hanahan method (Cohen, S.N. et al., Proc. Natl. Acac. Sd. USA, 9:2110-2114(1973); and Hanahan, D., J. MoI. Biol., 166:557-580(1983)) and electrophoresis (Dower, WJ. et al., Nucleic. Acids Res., 16:6127-6145(1988)) can be used for transformation. Also, in case of using eukaryotic cells as host, microinjection (Capecchi, M. R., Cell, 22:479(1980)), calcium phosphate precipitation (Graham, F.L. et al., Virology, 52:456(1973)), electrophoresis (Neumann, E. et al., EMBO 1, 1:841(1982)), liposome-mediated transfection (Wong, T.K. et al., Gene, 10:87(1980)), DEAE-dextran treatment (Gopal, MoI. Cell Biol., 5:1188-1190(1985)), and particle bombardment (Yang et al., Proc. Natl. Acad. ScL, 87:9568-9572(1990)) can be use for transformation.

Expression vectors in host cells express peptides or proteins of interest. For

example, if the expression vector carries lac promoter, the induction of expression can be performed by treatment of IPTG to host cells.

According to a preferred embodiment, the trasnformant of this invention is prokaryotic, most preferably, E coli.

In still further aspect of this invention, there is provided a method for producing a recombinant peptide or protein, which comprises the steps of: (a) transforming a host cell with the vector comprising a nucleotide sequence encoding the recombinant peptide or protein; (b) culturing the transformed host cell and expressing the recombinant peptide or protein; and (c) preparing the expressed the recombinant peptide or protein.

According to a preferred embodiment, the vector used comprises an expression construct containing (i) the PAS factor mutant-encoding nucleotide sequence-(ii) a fusion partner protein-encoding nucleotide sequence-(iii) the recombinant peptide or protein-encoding nucleotide sequence.

In the present method, the culturing transformed cells may be carried out in accordance with conventional media used in the art.

For instance, where the transformed cells are prokaryotic (e.g., E coll), culturing may be carried out using LB (Luria-Bertani) media. Where the transformants are animal cells, culturing may be performed using Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)).

Various processes of culturing are well known to those skilled in the art as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001), which is incorporated herein by reference. The step (c) for preparing recombinant peptides or proteins expressed may be carried out either during cell culturing in continuous culture, or after the completion of cell culturing in batch culture.

According to a preferred embodiment, the isolation of recombinant peptides or proteins is performed by collecting fusion proteins in a purified form. For example, the recombinant peptides or proteins may be obtained in a purified form by ammonium sulfate precipitation, an ultrafiltration with defined molecular weight cut- off value and/or various chromatography (designed for purification dependent upon size, charge, hydrophobicity and affinity).

According to a preferred embodiment, the collection of recombinant peptides or proteins is carried out by affinity chromatography. For example, where the recombinant peptides or proteins are fused to GST or 6X His, they may be obtained in a purified form using glutathione-binding resins or Ni-NTA His-binding resins, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents a ribbon diagram showing a three dimensional structure of the wild type PAS (Plasmid Acromobacter Secretion) factor. The diagram was prepared using PyMOL program available from http://pymol.sourceforqe.net/.

Fig. 2 represents distribution of electrostatic surface potential of the wild type PAS factor. The hydrophobic patch (indicated by dotted circle) was determined to interact with cell membrane. Fig. 3 represents the construction of the mutated PAS fusion vector. GST, MCS,

Kan ^R , Lad and P ₇₇ represent glutathione S-transferase gene, multiple cloning site, kanamycin resistant gene, Lad coding sequence and T7 promoter, respectively.

Fig. 4a is a gel photograph demonstrating the successful expression of calcipressin by the present vector. Fig. 4b is a gel photograph demonstrating the successful expression of ryanodine receptor by the present vector. 1, molecular marker; 2, conventional vector pET29a, before induction of expression; 3, conventional vector pET29a, after induction of expression; 4, conventional vector His-TEV, before induction of expression; 5, conventional vector His-TEV, after

induction of expression; 6, conventional vector Trx-TEV, before induction of expression; 7, conventional vector Trx-TEV, after induction of expression; 8, conventional vector Nus-TEV, before induction of expression; 9, conventional vector Nus-TEV, after induction of expression; 10, conventional vector GST-TEV, before induction of expression; 11, conventional vector GST-TEV, after induction of expression; 12, the wild type PAS expression vector, before induction of expression; 13, the wild type PAS expression vector, after induction of expression; 14 ^* , the mutated PAS expression vector, before induction of expression; 15 ^* , the mutated PAS expression vector, after induction of expression. Arrows denote expressed calcipressin and ryanodine receptor.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES Cells and Plasmids

The features of cells and plasmids used in these experiments are summarized in Table 1: TABLE 1

Escherichia coli cells used were cultured in LB (Luria Bertani) media (Merk,

Inc.). Following culture, cells were stored with 20% glycerol in -70 ⁰ C deep freezer.

EXAMPLE 1: Isolation of PAS Factor from Vibrio vulnificus

Vibrio vulnificus was cultured in 2.5% NaCI heart infusion (HI) media and its genomic DNA molecules were isolated by sodium dodecyl sulfate (SDS)-protease method (Little, P.F.R (1987) DNA Cloning: A practical approach. IRL press 3:19-42).

The isolated genomic DNA molecules were digested with Sau3AI and resolved on an agarose gel (Amersham Inc.). The agarose gel containing DNA fragments ranging 0.5-1.0 kb sizes was incubated with β-agarase (New England Biolabs; NEB Inc.). Using QIAEX II gel extraction kit (Qiagen Inc.), the DNA fragments in 0.5-1.0 kb size were obtained for the agarose gel. The DNA fragments were introduced into pET30a vector (Novagen Inc.) and transformed into E. coli DH5α cells, followed by culturing. From colonies generated, plasmid library DNA molecules were isolated. The isolated plasmid library DNA molecules were transformed into E cσ// BL21(DE3) (Novagen Inc.) and then expressed.

Antibodies specifically binding to Vibrio vulnificus were prepared using plasma samples from vibrio septicemia patients and proteins of Vibrio vulnificus expressed in E coli BL21(DE3) and then Colony Western blotting analysis was performed using antibodies and colonies. The expression library prepared was serially diluted, plated at a density of about 500 colonies/plate on plating media and cultured overnight. Afterwards, using sterilized velvet, replica plating culture was carried out on media containing isopropyl-β-D-thiogalactoside (IPTG) for 5 hr and the expression of antigens was induced. The culture plates were exposed to chloroform vapor for 15 min for cell lysis and proteins were transferred to nitrocellulose filter paper for 15 min. The nitrocellulose filter paper was incubated overnight with 5% non-fat milk, 0.5% Tween 20 and phosphate buffer (pH 7.2) in 4 ⁰ C refrigerator for blocking and then incubated with patient serum (diluted 1:5000) as a primary antibody for 1 hr at room temperature. Afterwards, the filter paper was incubated with peroxidase-

conjugated anti-human antibody as a secondary antibody and analyzed using ELC chemiluminescence kit (Amresham Co.). Twelve clones showing reactive colonies were selected.

For twelve colonies selected, DNA sequences were revealed using ABI Prism 377 automatic DNA sequencer (Perkin-Elmer Applied Biosystems Inc.).

Among several antigen genes analyzed by DNA sequencing, a novel PAS factor gene was selected. It was elucidated that the DNA sequence of the PAS factor gene had the nucleotide sequence set forth in SEQ ID NO:1 except that nucleotides 106-108 are gat.

EXAMPLE 2: Preparation of Expression Vectors Using PAS Factor

Using the PAS factor gene as templates cloned into pET30a vector, the PAS factor gene (231 bp) containing Nde l recognition site at its both ends was amplified.

For amplification, the sense primer 5'-GGTCATATGAAAGCCCTC (underline, Nde I recognition site) and antisense primer 5'-GCCCATATGGTGTTCAGG were used. The

PCR amplification was performed using reactants containing 100 ng template, 0.2 pM primers, 0.25 mM dNTPs and two units of pfu DNA polymerase (Stratagene Inc.).

The PCR reactions were conducted under the following thermal conditions: 5 min at 94 ⁰ C followed by 25 cycles of 30 sec at 94 ⁰ C , 30 sec at 5O ⁰ C, and 1 min at 72 ⁰ C; followed by a 10 min final extension at 72 ⁰ C. Amplified PAS genes with a size of 231 bp were purified using QIAEX II gel extraction kit (Qiagen Inc.).

The GST-Tag vector (pET41a) and PAS gene amplified were digested with Nde I (NEB Inc.) and purified. The PAS gene purified was cloned into pET41a vector to prepare a novel expression PAS fusion vector, PAS-GST-Tag vector. The PAS-GST- Tag vector contains a main skeleton originated from pET41a plasmid, the PAS factor gene, GST (glutathione S-transferase) tag, protease TEV recognition site and a multiple cloning site (MCS) for foreign DNA cloning in order.

EXAMPLE 3: Structural Analysis of PAS Factor

PAS factor crystals were grown using the hanging-drop, vapor-diffusion method. The best crystals were obtained from drops prepared by mixing 2 μl of protein solution (11.8 mg/ml of PAS factor in 20 mM Hepes-NaOH, pH 7.5, 150 mM KCI) and 2 μl of reservoir solution (0.1 M Tris-HCL, pH 8.5, 22%(w/v) PEG 4000, 0.2 M MgCI ₂ ). Large crystals were grown to a maximal size of 0.2 mm x 0.2 mm x 0.1 mm over one week. Successful flash-freezing was achieved when the crystals were transferred directly from the drop of reservoir solution containing 20 %(v/v) glycerol. Because bromine is a convenient anomalous scatterer for MAD (multiwavelength anomalous dispersion) phasing, a bromine MAD data set was collected from a single crystal soaked for 1 hr in cryoprotection solution containing 1 M NaBr. The MAD data set was then collected to 1.9 A resolution using an ADSC Quantum 4R CCD detector at beamline BL-18B at the Photon Factory, Japan. Data sets were processed and scaled with the program HKL2000. The structure of PAS factor was determined by MAD using bromine as an anomalous scatterer. The positions of five bromine groups in the asymmetric unit were located and refined using the program SOLVE (Terwilliger, T. C. & Berendzen, J. (1999). Automated MAD and MIR structure solution. Acta Crystallog. sect. D, 55, 849-861). The initial phases were improved by solvent flattening using the program RESOLVE (Terwilliger, T. C. (2000). Maximum likelihood density modification. Acta Crystallog. sect. D, 56, 965-972). The resultant map was readily interpretable, and model building proceeded using the program O (Jones, T. A., et al. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallog. sect. A, 47, 110-119), after which the initial model was refined using the program CNS (Brunger, A.T. et al., (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. sect. D, 54, 905-921). A manual rebuilding step was performed followed by a few cycles of conjugate gradient least-squares

refinement using SHELXL (Sheldrick, G. & Schneider, T. (1997). SHELXL: highresolution refinement. Methods Enzymol. 277, 319-343). A final round of anisotropic refinement with REFMAC5 (Murshudov, G. N., et al., (1999). Efficient anisotropic refinement of Macromolecular structures using FFT. Acta Crystallog. sect. D, 55, 247-255) was also preformed. The final crystallographic R value for the PAS factor model, using data from 25 A to 1.8 A was 19.9% (R _free = 23.2%).

We analyzed the three dimensional structure of the PAS factor by X-ray crystallography, and found that the PAS factor have a typical five-helix bundle structure (Fig. 1). The five-helix bundle structure is a compact domain consisting of five α helices in parallel. Hydrophobic residues are buried inside the helical bundle, while hydrophilic residues are exposed on the surface of the PAS factor. The inner part of bundle structures is so compacted that one molecule of water cannot be incorporated into. As shown in Fig. 2 representing electrostatic surface potential distribution of the PAS factor, the third helix was determined to interact with cell membrane (analyzed by tryptophan fluorescence spectroscopy).

These results urge us to reason that a substitution in which a hydrophilic amino acid residue, aspartic acid positioned at amino acid 36 of the PAS factor is replaced with a hydrophobic residue, proline, could contribute to not only enhancement of binding strength of the PAS factor to cell membrane but also improvement in its chaperone function. In addition, it could be appreciated that the substitution would result in significant improvement in PAS fusion expression vectors.

EXAMPLE 4: Preparation of Mutant PAS Fusion Vector by Site-Directed Mutagenesis To replace an aspartic acid residue positioned at amino acid 36 of the wild

PAS factor by a proline residue, two primers (PAS-F-D36P and PAS-R-D36P) were constructed for site-directed mutagenesis: PAS-F-D36P, 5'-

GMCAGTTGGATTTGCCTTTTCCAATGCAACTGGCTTTGTATGC-S'; and PAS-R-D36P,

5'-GCATACAAAGCCAGTTGCATTGGAAAAGGCAAATCCAACTGTTC. The underlined letters are bases mutated for substitution of aspartic acid with proline.

Using the PAS-GST-Tag vector as templates constructed in Example 2, mPAS- GST-Tag vector with a size of 5.8 kb containing mutated PAS factor was prepared and amplified. The PCR amplification was performed using 50 μl reactant containing 100 ng template, 0.2 pM primers, 0.25 mM dNTPs and two units of pfu DNA polymerase (Stratagene Inc.). The PCR reactions were conducted under the following thermal conditions: 5 min at 94 ⁰ C followed by 25 cycles of 30 sec at 94 ⁰ C , 30 sec at 5O ⁰ C, and 1 min at 72 ⁰ C; followed by a 10 min final extension at 72 ⁰ C. The amplified produces were resolved on 1% agarose gel. The mPAS-GST-Tag vector with a size of 5.8 kb amplified was purified using QIAEX II gel extraction kit (Qiagen Inc.).

The purified products were digested with Dpnl (NEB Inc.) and then purified using QIAEX II gel extraction kit (Qiagen Inc.). The construction of the mutated PAS fusion vector finally purified is represented in Rg. 3. The sequence of the mutated PAS fusion vector was analyzed using ABI Prism 377 automatic DNA sequencer (Perkin-Elmer Applied Biosystems Inc.), elucidating that the aspartic acid residue positioned at amino acid 36 is successfully substituted with a proline residue.

EXAMPLE 5: Evaluation on Performance of Mutated PAS Fusion Vector

To evaluate performance of the mutated PAS fusion vector of this invention, five conventional vectors, pET29a (Novagen), His-TEV vector [vector containing a TEV recognition site at upstream of a multiple cloning site in pET28a vector (Novagen)], Nus-TEV vector [vector containing a NusA-Tag encoding sequence and a TEV recognition site at upstream of a multiple cloning site in pET28a vector (Novagen)], Trx-TEV vector [vector containing a Trx-Tag encoding sequence and a TEV recognition site at upstream of a multiple cloning site in pET28a vector (Novagen)] and GST-TEV vector [vector containing a GST-Tag encoding sequence

and a TEV recognition site at upstream of a multiple cloning site in pET41a vector (Novagen)], were used to express proteins of interest. In addition, the protein expression was performed using the wild type PAS fusion vector. The His-TEV vector, Nus-TEV vector, Trx-TEV vector and GST-TEV vector are also described in more detail in Korean Pat. Appln. No. 2005-0051893.

The expression potency of vectors was tested for one of transmembrane proteins, ryanodine receptor involved in human muscle relaxation/contraction and human-originated calcipressin. These proteins are not expressed in both conventional vectors and wild type PAS fusion vectors. Five conventional vector including pET29a, His-TEV vector, Nus-TEV vector,

Trx-TEV vector and GST-TEV vector, the wild type PAS fusion vector, the mutated PAS fusion vector, and genes encoding proteins of interest were digested with Hind III and Xho I (NEB), and each gene digested was then cloned into vectors. Cloned genes were transformed into E.coli BL21(DE3) by the calcium chloride method and cells were cultured in 3 ml of LB liquid media containing 25 μg/ml kanamycin over 16 hr at 37 ⁰ C. 50 ml of liquid media inoculated with aliquot of the media (500 μl) were cultured with shaking of 130 rpm at 37 ⁰ C, and added with 0.25 mM IPTG until their optical density at 600 nm reached 0.45. Following 6-hr culturing, an aliquot of culture media was resolved by 12% polyacrylamide gel electrophoresis to determine the expression level of proteins (Figs. 4a and 4b).

As shown in lanes 1-11 of Figs. 4a and 4b, five conventional vectors could not express ryanodine receptor and calcipressin. In addition, the wild type PAS fusion vector also could not express ryanodine receptor and calcipressin. In contrast, the mutated PAS fusion vector of this invention successfully induced the expression of ryanodine receptor and calcipressin as shown in lane 15.

As results, it could be understood that the mutated PAS vector of this invention has much better expression potency than conventional vectors and non- mutated PAS fusion vectors.

The vectors comprising the PAS factor mutant lead to significant enhancement in the expression of peptides or proteins fused into them. Furthermore, the PAS mutant makes it possible to express transmembrane proteins such as ryanodine receptor and human-originated proteins such as calcipressin which have been reported to be scarcely expressed.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.