WO2010082804A2

WO2010082804A2 - Method for producing physiologically active protein or peptide using immunoglobulin fragment

Info

Publication number: WO2010082804A2
Application number: PCT/KR2010/000342
Authority: WO
Inventors: Jin-Sun Kim; Sung Youb Jung; Jong-Soo Lee; Byung Sun Lee; Se Chang Kwon; Gwan Sun Lee
Original assignee: Hanmi Pharm. Co., Ltd.
Priority date: 2009-01-19
Filing date: 2010-01-19
Publication date: 2010-07-22
Also published as: WO2010082804A3; TW201031752A; KR20100084996A; AR075029A1

Abstract

The present invention provides a method for mass-producing a physiologically active protein or peptide using a fusion protein composed of the physiologically active protein or peptide and an immunoglobulin fragment. The present invention also provides such a fusion protein, a DNA encoding the fusion protein, an expression vector comprising the DNA, and a microorganism transformed with the expression vector.

Description

METHOD FOR PRODUCING PHYSIOLOGICALLY ACTIVE PROTEIN OR PEPTIDE USING IMMUNOGLOBULIN FRAGMENT

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Various peptides have emerged as promising therapeutics that can partly replace the existing protein therapeutics, and they are generally produced by various chemical synthetic methods. However, the chemical synthetic methods have problems such as the generation of by-products and high production costs. Recent advances in recombinant DNA technologies have made it possible to mass-produce peptides having about 50 amino acid residues or proteins composed of more amino acid residues by employing a genetic recombination method at a reasonable cost. Such genetic recombination method, however, has a problem in that it is difficult to obtain a low-molecular weight peptide such as some hormones and cytokines in the natural form in host cells, because the peptide is apt to be modified after the expression, e.g., due to the digestion by proteases of the host cells. Further, most of the proteins and peptides expressed in bacterial cells have an additional methionine residue in their amino terminus, and such methionine residue not only affects the activity and stability of the proteins and peptides, but may also induce adverse immune responses in a subject treated with such therapeutic proteins or peptides.

The above problems may be solved by recombinantly producing a desired protein or peptide in the form of a fusion protein with a specific protein. Generally, many heterogenous proteins and peptides may be produced in baterial or animal cells as proteins fused with one of highly expressible fusion partner proteins, e.g., LacZ, GST, thfB, bla, E. coli maltose-binding protein (MBP) and E. coli thioredoxin, by employing a fused gene consisting of a gene for the target protein or peptide and a gene for the highly expressible protein. This fusion protein expression method has advantages in that: it is possible to mass-produce the desired protein or peptide owing to the high expressibility of the fusion partner protein; the stability and solubility of the desired protein or peptide become enhanced; and the fusion protein can be separated and purified by exploiting the affinity thereof.

A protein or peptide produced in the form of a fusion protein can not be used for therapeutic use as is and it is necessary to isolate and recover the desired protein and peptide therefrom in a pure form. For this purpose, a genetically engineered protease cleavage site is inserted between the partners of a fusion protein and the produced fusion protein is cleaved with a protease to isolate the desired protein or peptide. However, this method can be used limitedly.

The present inventors have endeavored to develop a method for the mass- production of a physiologically active protein or peptide by employing a fusion protein, and have achieved the method of the present invention comprising the steps of: a) introducing a DNA coding for a fusion protein composed of an immunoglobulin fragment and a physiologically active protein or peptide into a cell, and culturing the cell; and b) isolating the fusion protein from the resulting cell culture and separating the physiologically active protein or peptide from the fusion protein.

SUMMARY QF THE INVENTION

Accordingly, it is an object of the present invention to provide a method for mass-producing a physiologically active protein or peptide comprising the steps of: a) introducing a DNA coding for a fusion protein composed of an immunoglobulin fragment and a physiologically active protein or peptide into a cell, and culturing the cell; and b) isolating the fusion protein from the resulting cell culture and separating the physiologically active protein or peptide from the fusion protein. It is another object of the present invention to provide such a fusion protein, a DNA encoding the fusion protein, an expression vector comprising the DNA and a microorganism transformed with the expression vector.

It is a further object of the present invention to provide a method for mass-producing a physiologically active protein or peptide using an immunoglobulin fragment as a fusion partner.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

Fig. 1 : the cloning scheme for fusing the immunoglobulin fragment with ubiquitin-specific cleavage sequence (Example 1-1); Fig. 2: the cloning scheme for fusing the protein and peptide with the pFUBPTHl-84 expression vector (Example 1-3);

Fig. 3: the cloning scheme for fusing the immunoglobulin fragment which contains EK-specific cleavage sequence with IGF-I peptide (Example 1-4);

Fig. 4: the preparation scheme of the expression vector by way of in- frame fusion with the immunoglobulin fragment and fuzeon peptide (Example 1-

5);

Fig. 5: SDS-PAGE analysis of glucagon peptide collected from the fusion protein by ubiquitin hydrolyse (Example 5); and

Fig. 6: SDS-PAGE analysis of glucagon (A)₅ IGF-I(B), PTH 1~84(C) and fuzeon (D) peptides (Example 6). DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a method for mass-producing a physiologically active protein or peptide comprising the steps of: a) introducing a DNA coding for a fusion protein composed of an immunoglobulin fragment and a physiologically active protein or peptide into a cell, and culturing the cell; and b) isolating the fusion protein from the resulting cell culture and separating the physiologically active protein or peptide from the fusion protein. In the present invention, the fusion protein comprises the immunoglobulin fragment and the physiologically active protein or peptide, which are operately linked to each other. The fusion protein further comprises an amino acid cleavage sequence (chemical- or enzyme-specific cleavage sequence) disposed between the immunoglobulin fragment and the physiologically active protein or peptide, which enables certain chemicals or enzymes to specifically recognize and cleave it.

In one aspect of the present invention, the method for mass-producing a physiologically active protein or peptide comprises the steps of: 1) preparing an expression vector comprising a DNA encoding a fusion protein consisting of an immunoglobulin fragment, an enzyme-specific cleavage sequence, and a physiologically active protein or peptide; 2) transforming a microorganism with the expression vector to obtain a transformed cell; 3) culturing the transformed cell to express a fusion protein; 4) recovering and refolding the fusion protein; and 5) collecting the physiologically active protein or peptide from the fusion protein using an enzyme.

In the present invention, the "immunoglobulin fragment" used as a fusion partner may be selected from the group consisting of the constant region of IgG, IgA, IgE, IgM, or IgD of human, mouse, pig, rabbit, or rat originated, and a combination or a hybrid thereof. Preferably, the immunoglobulin fragment may be selected from the group consisting of the constant region of IgGl, IgG2, IgG3, or IgG4, and a combination or a hybrid thereof, more preferably, the constant region of IgG4.

The term "combination" as used herein, means that a polypeptide encoding a single-chain immunoglobulin constant region binds to another one from different origin, to form a dimer or multimer. The term "hybrid" as used herein, means that there exist at least two immunoglobulin constant region sequences from different origin within a single-chain immunoglobulin constant region.

The immunoglobulin fragment may be a whole or fragment of at least one domain selected from the group consisting of the constant region of heavy chain including CHl domain, CH2 domain, CH3 domain and CH4 domain; and the constant region of light chain including CL domain. For example, the domain may include CHl domain, CH2 domain, CH3 domain, or CH4 domain; CHl and CH2 domains; CH2 and CH3 domains; and CHl, CH2 and CH3 domains, and the arrangement of domains is not limited. Preferably, the immunoglobulin fragment may comprise CHl, CH2 and

CH3 domains, and may further comprise CH4 domain (in case of IgM). Also, the immunoglobulin fragment may be a Fc fragment of the constant region of heavy chain, or a Fc fragment derivative, which may comprise a whole or fragment of hinge region. The hinge region may be a wild-type or be modified by deletion, addition and conservative or non-conservative substitution of amino acid residues in various sites therein.

The term "Fc fragment derivative" as used herein, refers to any modified immunoglobulin fragments from the native Fc fragment due to a modification of at least one amino acid residues. Fc fragment derivative can be prepared by genetic engineering known in the art.

In a preferred embodiment of the present invention, the immunoglobulin fragment comprises CHl, CH2 and CH3 domains of the constant region of IgG4. In another embodiment of the present invention, the immunoglobulin fragment may be a Fc fragment consisting of IgG4 hinge region, and CH2 and CH3 domains of the constant region of IgG4, or a derivative thereof. Preferably, the immunoglobulin fragment is may be encoded by the nucleotide sequence of SEQ ID NO: 1. In the present invention, "physiologically active protein or peptide" may be a therapeutically useful protein or peptide, but has a very low expression level or has difficulty in the preparation thereof.

Specifically, the physiologically active protein or peptide may be selected from the group consisting of blood factor, digestive hormone, adrenocorticotropic hormone, thyroid hormone, intestinal hormone, cytokine, enzyme, growth factor, neuropeptide, hypophyseotropic hormone, hypophysiotropic hormone, anti-viral peptide, and a non-native peptide derivative thereof retaining physiologically active property. Preferably, the physiologically active protein or peptide may be selected from the group consisting of erythropoietin, GM-CSF (granulocyte macrophage- colony stimulating factor), amylin, glucagon, insulin, somatostatin, PYY (peptide YY), NPY (neuropeptide Y), angiotensin, bradykinin, calcitonin, corticotropin, eledoisin, gastrin, leptin, oxytocin, vasopressin, LH (luteinizing hormone), prolactin, FSH (follicle stimulating hormone), PTH (parathyroid hormone), secretin, sermorelin, hGH (human growth hormone), growth hormone-releasing peptide, G-CSFs (granulocyte colony stimulating factor), interferons, interleukins, prolactin-releasing peptide, orexin, thyroid-releasing peptide, cholecystokinin, gastrin-inhibiting peptide, calmodulin, gastrin-releasing peptide, motilin, vasoactive intestinal peptide, ANP(atrial natriuretic peptide), BNP (barin natriuretic peptide), CNP (C-type natriuretic peptide), neurokinin A, neuromedin, renin, endothelin, sarafotoxin peptide, carsomorphin peptide, dermorphin, dynorphin, endorphin, enkepalin, T cell factor, tumor necrosis factor, tumor necrosis factor receptor, urokinase receptor, tumor inhibitory factor, collagenase inhibitor, thymopoietin, thymulin, thymopentin, tymosin, thymic humoral factor, adrenomodullin, allatostatin, amyloid beta-protein fragment, antimicrobial peptide, antioxidant peptide, bombesin, osteocalcin, CART peptide, E-selectin, ICAM-I, VCAM-I, leucokine, kringle-5, laminin, inhibin, galanin, fibronectin, pancreastatin, and fuzeon. In addition, derivatives modified from the native forms of the physiologically active polypeptides by substitution, insertion or deletion of some amino acid residues thereof, are also included within the scope of the physiologically active polypeptides of the present invention as long as it has a physiological function, activity or stability substantially identical or improved compared to native forms of the physiologically active polypeptides. These proteins or peptides may be produced in a high yield according to the present invention and be effectively used as therapeutics.

The term "chemical- or enzyme-specific cleavage sequence", as used herein, refers to one or more consecutive amino acid sequences that can be specifically recognized and digested by certain chemicals or enzymes. For instance, it was reported that cyanogen-bromide cleaves at single methionine amino acid cleavage sites, and that trypsin cleaves at single arginine or lysine or double repeated arginine-arginine or lysine-lysine cleavage sites (U.S. Patent NO. 6,010,883). Further, enterokinase recognizes the amino acid sequence of Asp- Asp-Asp-Asp-Lys (SEQ ID NO: 57) to cleave a peptide chain at Lys position, Xa protease factor recognizes the amino acid sequence of Ile-Glu-Asp-Gly-Argj (SEQ ID NO: 58), trombin recognizes the amino acid sequence of Leu-Val-Pro- Arg|Gly-Ser (SEQ ID NO: 59), TEV protease recognizes the amino acid sequence of Gru-Asn-Leu-Tyr-Phe-GlnjGly (SEQ ID NO: 60), and PreScissionTM protease recognizes the amino acid sequence of Leu-Glu-Val-Leu- Phe-GlnjGly-Pro (SEQ ID NO: 61) (Raymond. C. Steven., Structure 2000, vol 8, No. 9, 177-185), wherein the symbol of "J," refers to a cleavage site. Moreover, signal peptidase cleaves a signal sequence, and ubiquitin C-terminal hydrolase L3 cleaves the amino acid sequence by recognizing three cleavage sites as below.

MOIF¹³VKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPD^OORLIFA GKOLEDGRTLSDY NIOKESTL³⁾HLVLRLRGGj(Keith. D. Wilkins., J. MoI. Biol (1999)291, 1067-1077) (SEQ ID NO: 62)

In the present invention, the chemical- or enzyme-specific cleavage sequence may be specifically recognized and cleaved by a chemical or an enzyme in order to isolate a target physiologically active protein or peptide. The chemical- or enzyme-specific cleavage sequence may be preferably recognized by a protein selected from the group consisting of trypsin, trombin, TEV protease, PreScission protease, enterokinase and ubiquitin hydrolase, more preferably, enterokinase or ubiquitin C-terminal hydrolase L3, most preferably, ubiquitin C- terminal hydrolase L3.

The ubiquitin hydrolase used in the present invention can specifically recognize a tertiary structure of proteins or peptides and cleave C-terminal thereof, to obtain the desired proteins or peptides. The ubiquitin hydrolase also makes it possible to attain a high level of expression of the target protein or peptide since size itself is small (Bull. Korean Chem. Soc. 2007, Vol. 28, No. 9), and even shows a high cleavage yield under the high concentrated salt conditions. The term "fusion protein composed of an immunoglobulin fragment and a physiologically active protein or peptide" or "fusion protein", as used herein, refers to one or more polypeptides that are linked through a peptide bond to foπn a single-chain polypeptide. The fusion protein may be prepared by being translated into a single polypeptide from a genetically recombined nucleotide sequence fused with one or more genes including an immunoglobulin gene.

In a preferred embodiment, the fusion protein composed of an immunoglobulin fragment and a physiologically active protein or peptide may be prepared by the expression of the recombined nucleotide sequence fused with a nucleotide sequence encoding an immunoglobulin fragment, and a chemical- or emzyme-specific sequence encoding a target protein or peptide.

Fusion partners are used i) to yield a high expression of a protein or peptide whose original expression level is low and artificial synthesis is difficult; ii) to induce the secretion of the expressed protein or peptide into a medium or the outside of the cytosol; iii) to increase the solubility and stability of the expressed protein or peptide; and iv) to easily purify the expressed protein or peptide. As the fusion partner, His-Tag, T7-Tag, S-tag, Flag peptide, ubiquitin, NusA, chloramphenicol acetyltransferase, streptococcal G protein, dihydrofolate reductase, cellulose binding domains (CBD's), galactose binding protein, calmodulin binding protein (CBP), hemagglutinin influenza virus (HAI), green fluorescent protein (GFP), chitin binding domain, ompT (outer membrane proteinT), ompA (outer membrane proteinA), pelB, DsbA (disulfide-bond formation facilitator A), DsbC (disulfide-bond formation facilitator C), HSB-tag, polyarginine, polycysteine, polyphenylalanine, c-myc, T7genlO, KSI, LacZ (β- galactosidase), glutathione-S-transferase, thfB, bla, E. coli MBP, E. coli thioredoxin and the like are widely used.

In general, LacZ (β-galactosidase), glutathione-S-transferase, thfB, bla and E. coli MBP, which are frequently used as a fusion partner, have been employed to introduce water-solubility into an expressed fusion protein or to facilitate its purification. However, since proteins are frequently expressed as inclusion bodies depending on a fused protein or peptide, the application of the fusion partner is limited only to some proteins or peptides of suitable characteristics. In addition, only a fusion protein expressed in a water-soluble form can be purified by affinity purification method, and thus the fusion protein expressed as inclusion bodies is difficult for purification and refolding.

Whereas, the fusion protein of the present invention can be easily expressed in a water-soluble or insoluble inclusion body form in a host cell based on the expression strategy. Particularly, the fusion protein of the present invention enables a protein expressed as inclusion bodies to refold easily so that it can be employed in the expression of most proteins and peptides. Further, it can be advantageously subjected to both purification by protein A affinity column using the affinity of immunoglobulin and purification by ion exchange resin.

The immunoglobulin fragment used as a fusion partner in the present invention makes it possible to attain a high level of expression of the target protein or peptide to be fused in a water-soluble or insoluble inclusion body form to purity them easily. Specifically, the immunoglobulin fragment used as a fusion partner in the present invention shows high protein expression level with high efficiency in a microbial host cells including E. coli, and the conjugated protein or peptide of interest may be also a novel fusion partner being capable of providing high protein expression level.

In accordance with another aspect of the present invention, there is provided a fusion protein comprising an immunoglobulin fragment and a physiologically active protein or peptide, having the amino acid sequence of SEQ ID NO: 3.

In accordance with a further aspect of the present invention, there is provided a DNA encoding the fusion protein, an expression vector comprising the DNA and a microorganism transformed with the expression vector.

In a preferred embodiment of the present invention, the microorganism transformed with the expression vector is selected from the group consisting of E. coli HMFOOl (Accession NO: KCCM-10980P), HMF002, HMF003 (Accession

NO: KCCM-10981P), HMF004, HMF005, HMF006, HMF007, HMF008,

HMF009, HMFOO 10, HMFOO 11 , and HMFOO 12. The microorganism HMFOO 1 and HMF003 have been deposited at Korea Culture Center of Microorganism (KCCM) on 15^th January, 2009 under the Accession numbers of KCCM-10980P and KCCM-1098 IP, respectively.

In accordance with a still further aspect of the present invention, there is provided the method for mass-producing a physiologically active protein or peptide by using an immunoglobulin fragment as a fusion partner.

The following Examples are intended to further illustrate the present invention without limiting its scope.

Example 1: Construction of fusion protein expression vector

1-1. Construction of immunoglobulin fragment-ubiquitin fusion protein expression vector

An expression vector encoding a fusion protein composed of an immunoglobulin fragment and ubiquitin was constructed. The expression vector comprises a DNA encoding the human immunoglobulin Fc fragment of SEQ ID NO: 1 consisting of IgG4 hinge region, and CH2 and CH3 domains of the constant region of heavy chain of IgG4, and a DNA encoding the ubiquitin of SEQ ID NO: 2 comprising an enzyme-specific cleavage sequence recognizable by protease (ubiquitin), which are operately linked to each other.

In order to construct the expression vector for fusion protein, the DNA encoding the human immunoglobulin fragment was synthesized based on the blood cDNA library of Clontech laboratories Inc. A forward oligonucleotide of SEQ ID NO: 4 was synthesized to include ATG start codon and Ndel restriction enzyme recognition sites, and a reverse oligonucleotide of SEQ ID NO: 5 was synthesized without inserting a restriction enzyme site at the 5' terminal for in- frame fusion with ubiquitin-coding sequence. The DNA encoding the immunoglobulin fragment thus obtained was amplified with PCR. The PCR was conducted under the condition of 30 cycles of amplification for 30 sec at 60⁰C (annealing); and 50 sec at 68 ^°C (extension). The DNA encoding the ubiquitin comprising an enzyme recognition site was synthesized from cDNA library. For in-frame fusion with the immunoglobulin fragment, the forward oligonucleotide of SEQ ID NO: 6 was synthesized to include only a sequence encoding ubiquitin, and the reverse oligonucleotide of SEQ ID NO: 7 was synthesized to include MSCI and BamHI restriction enzyme recognition sites. The DNA encoding ubiquitin thus obtained was amplified with PCR. The PCR was conducted under the condition of 30 cycles of amplification for 30 sec at 60 °C (annealing); and 30 sec at 68 ^°C (extension). The amplified DNAs of the immunoglobulin fragment (666bp) and ubiquitin (228bp) were cloned into vector pET22b (Novagen). Specifically, vector pET22b was digested with Ndel and BamHI to delete signal sequences. The DNAs of the immunoglobulin fragment and ubiquitin obtained by PCR were digested with restriction enzymes of Ndel and BamHI, respectively, and the resulting DNAs were cloned into vector pET22b using T4 DNA ligase. The expression vector thus obtained was designated "pCarrierA-UB" fusion protein expression vector and the cloning procedure thereof is shown in Fig. 1. The pCarrierA-UB fusion protein expression vector comprises the nucleotide sequence of SEQ ID NO: 3 under the control of T7 promoter and expressed the fusion protein in the form of an inclusion body in a host cell.

1-2. Preparation of peptide

Glucagon (29 a.a), Salmon calcitonin (32 a.a), PYY3-36 (34 a.a), PTH (1-84) (84 a.a), PTH (1-34) (34 a.a), IGF-I (70 a.a), leptin (167 a.a) and fuzeon (36 a.a) peptides were synthesized by LCR (ligation chain reaction) or PCR using oligonucleotides comprising respective peptide sequences.

Specifically, 10 pmol of oligonuleotides comprising respective peptide sequences was added to a reaction tube containing a mixture of 0.1 mM dNTP, 1OX reaction buffer and 1 unit of pfu polymerase, and the resulting mixture was subjected to LCR or PCR using verity PCR cycler (applied biosystem).

In the present invention, LCR was conducted under the condition of 30 cycles of amplification for 20 sec at 55⁰C (annealing); and 60 sec at 68 ^"C (extention), and PCR was conducted under the condition of 30 cycles of amplification for 30 sec at 60 °C (annealing); and 30 sec at 68 °C (extention).

a) Preparation of glucagon peptide

The glucagon peptides were prepared by LCR using oligonucleotides of

SEQ ID NOs: 8 to 14. At this time, to facilitate the in-frame fusion procedure, a base of "C" was added to 5' forward oligonucleotide of SEQ ID NO: 8, and Sail restriction enzyme recognition site was inserted into 3' reverse oligonucleotide of SEQ ID NO: 14.

b) Preparation of salmon calcitonin peptide

The salmon calcitonin peptides were prepared by LCR using oligonucleotides of SEQ ID NOs: 15 to 21. At this time, to facilitate the in- frame fusion procedure, a base of "C" was added to 5' forward oligonucleotide of SEQ ID NO: 15, and Sail restriction enzyme recognition site was inserted into 3' reverse oligonucleotide of SEQ ID NO: 21.

c) Preparation of PYY (peptide YY)(3~36) peptide

The PYY (3~36) peptides were prepared by LCR using oligonucleotides of SEQ ID NOs: 22 to 28. At this time, to facilitate the in-frame fusion procedure, a base of "C" was added to 5' forward oligonucleotide of SEQ ID NO:

22, and Sail restriction enzyme recognition site was inserted into 3' reverse oligonucleotide of SEQ ID NO: 28.

d) Preparation of PTH (parathormone)(l~84) peptide

The PTH (1-84) peptides were prepared by PCR using oligonucleotides of SEQ ID NOs: 29 and 30 prepared from human placenta cDNA library (OriGene technologies, Inc.). At this time, to facilitate the in-frame fusion procedure, a base of "C" was added to 5' forward oligonucleotide of SEQ ID NO: 29, and Sail restriction enzyme recognition site was inserted into 3' reverse oligonucleotide of SEQ ID NO: 30.

e) Preparation of PTH (1-34) peptide

The PTH (1~34) peptides were prepared by PCR using oligonucleotides of SEQ ID NOs: 31 and 32, and the PCR products of PTH (1-84) peptides as a template. At this time, to facilitate the in-frame fusion procedure, a base of "C" was added to 5' forward oligonucleotide of SEQ ID NO: 31, and Sail restriction enzyme recognition site was inserted into 3 ' reverse oligonucleotide of SEQ ID NO: 32.

f) Preparation of IGF- 1 peptide

The IGF (insulin like growth factor)- 1 peptides were prepared by PCR using oligonucleotides of SEQ ID NOs: 33 to 35 prepared from human placenta cDNA library (OriGene technologies, Inc.). At this time, to facilitate the in- frame fusion procedure, a base of "C" was added to 5' forward oligonucleotide of SEQ ID NO: 33, and Sail restriction enzyme recognition site was inserted into 3' reverse oligonucleotide of SEQ ID NO: 34.

g) Preparation of leptin peptide

The leptin peptides were prepared by PCR using oligonucleotides of SEQ ID NOs: 36 and 37 prepared from human leptin cDNA library (OriGene technologies, Inc.). At this time, to facilitate the in-frame fusion procedure, a base of "C" was added to 5' forward oligonucleotide of SEQ ID NO: 36, and Sail restriction enzyme recognition site was inserted into 3' reverse oligonucleotide of SEQ ID NO: 37. h) Preparation of fuzeon peptide

The fuzeon peptides were prepared by LCR using oligonucleotides of

SEQ ID NOs: 38 to 44. At this time, to facilitate the in-frame fusion procedure, a base of "C" was added to 5' forward oligonucleotide of SEQ ID NO: 38, and

Sail restriction enzyme recognition site was inserted into 3' reverse oligonucleotide of SEQ ID NO: 44.

1-3. Preparation of fusion protein of immunoglobulin fragment-ubiquitin(UB)- physiologically active protein or peptide

A fusion protein comprising the immunoglobulin fragment-UB fusion protein and a physiologically peptide was prepared by employing in-frame fusion between a expression vector of the immunoglobulin fragment-UB fusion protein and a DNA sequence encoding the peptide.

Specifically, the respective DNAs encoding each peptide (PTH 1~84, PTH1-34, Glucagon, sCalsitonin, IGF-I, PYY3-36 and Fuzeon) prepared in 1-2 above were digested with Sail restriction enzyme, and the resulting DNAs corresponding to a size of the respective peptides were collected (PTH 1~84: 252bp; PTH1-34: 102bp; Glucagon: 87bp; sCalsitonin: 96bp; IGF-I: 210bp; PYY3-36: 102bp and Fuzeon: 108bp).

Also, the immunoglobulin fragment-UB fusion protein expression vector (pCanϊerA-UB) was digested with restriction enzymes of MSCI and Sail, and the resulting expression vector was ligated with the DNA encoding any one of the peptides using T4 DNA ligase, to obtain expression vectors for expressing fusion protein of immunoglobulin fragment-UB -peptide.

The respective expression vectors thus obtained designated immunoglobulin fragment-UB-PTH(l~84)(pFUBPTHl-84), immunoglobulin fragment-UB-PTH(l~34)(pFUBPTHl-34), immunoglobulin fragment-UB -IGF- l)(pFUBIGF), immunoglobulin fragment-UB-sCalcitoninOpFUBCal), immunoglobulin fragment-UΕ-glucagon(pFUBGluca), immunoglobulin fragment-UB-PYY(3~36)(pFUBPYY3-36), immunoglobulin fragment-UB- leptin(pFUBLep) and immunoglobulin fragment-UB-fuzeon(pFUBFuzeon), respectively. The cloning procedure of one of the expression vectors, pFUBPTHl-84 is shown in Fig. 2. The expression vectors were expressed the fusion proteins in the form of an inclusion body in a host cell.

1-4. Preparation of fusion protein of immunoglobulin fragment-EK-IGF-1

An expression vector for expressing a fusion protein composed of an immunoglobulin fragment-EK and IGF-I peptide was constructed. The expression vector comprises a DNA encoding the human immunoglobulin Fc fragment of SEQ ID NO: 1 consisting of IgG4 hinge region, and CH2 and CH3 domains of the constant region of heavy chain of IgG4. 3' terminal of the immunoglobulin fragment-coding sequence was ligated with the 5' terminal of IGF-I peptide-coding sequence comprising an enzyme-specific cleavage sequence of SEQ ID NO: 63 recognizable by enterokinase (EK).

In order to contract the expression vector for fusion protein, the DNA encoding the human immunoglobulin fragment was synthesized based on the blood cDNA library of Clontech laboratories Inc. At this time, A forward oligonucleotide of SEQ ID NO: 45 was synthesized to include ATG start codon and Ndel restriction enzyme recognition site, and a reverse oligonucleotide of SEQ ID NO: 46 was synthesized to include an EK-specific cleavage sequence of SEQ ID NO: 63. A DNA comprising the immunoglobulin fragment and EK- specifϊc recognition site was prepared by PCR using the oligonucleotides. The PCR was conducted under the condition of 30 cycles of amplification for 30 sec at 60 "C (annealing); and 50 sec at 68 ^°C (extension).

A DNA encoding the IGF-I peptides was amplified by PCR using oligopeptides of SEQ ID NOs: 47 and 48. At this time, the EK-specific recognition site was inserted into 5' forward oligonucleotide of SEQ ID NO: 47, and BamHI restriction enzyme recognition site was inserted into 3' reverse oligonucleotide of SEQ ID NO: 48.

A fusion DNA of immunoglobulin fragment-EK-IGF-1 was synthesized by PCR using the DNAs of the immunoglobulin fragment-EK and IGF-I peptides. The fusion DNA thus obtained was digested with restriction enzymes of Ndel and BamHI, and the resulting fusion DNA was cloned into vector pET22b, which was previously digested with Ndel and BamHI to delete signal sequences, using T4 DNA ligase. The expression vector thus obtained was designated immunoglobulin fragment-EK-IGF-1 (pFEKIGF) and the cloning procedure thereof is shown in Fig. 3.

1-5. Preparation of fusion protein of immunoglobulin fragment-fuzeon, immunoglobulin fragment-fuzeon-fuzeon and immunoglobulin fragment-C34 fuzeon

1) Construction of immunoglobulin fragment-fuzeon expression vector

An expression vector for expressing a fusion protein composed of immunoglobulin fragment and fuzeon peptide was constructed. The expression vector comprises a DNA encoding the human immunoglobulin Fc fragment of SEQ ID NO: 1 consisting of IgG4 hinge region, and CH2 and CH3 domains of the constant region of heavy chain of IgG4. 3' terminal of the immunoglobulin fragment-coding sequence is ligated with 5' terminal of fuzeon peptide-coding sequence by in-frame fusion.

In order to construct the expression vector for the fusion protein, the DNA encoding the human immunoglobulin fragment was synthesized based on the blood cDNA library of Clontech laboratories Inc. At this time, a forward oligonucleotide of SEQ ID NO: 49 was synthesized to include ATG start codon and Ndel restriction enzyme recognition site, and a reverse oligonucleotide of SEQ ID NO: 50 was synthesized by inserting only "p" (meaning phosphate) and without inserting a restriction enzyme site at 5' terminal site for in-frame fusion with fuzeon pepetide-coding sequence. The DNA encoding the immunoglobulin fragment thus obtained was amplified with PCR. The PCR was conducted under the condition of 30 cycles of amplification for 30 sec at 60⁰C (annealing); and 50 sec at 68 ^°C (extension).

DNAs coding fuzeon peptide were synthesized using the DNA encoding immunoglobulin fragment-UB -fuzeon prepared in Example 1-3 as a template DNA. For in-frame fusion with the immunoglobulin fragment, "p" (meaning phosphate) was added to the forward oligonucleotide of SEQ ID NO: 51 and BamHI restriction enzyme recognition site was inserted into the reverse oligonucleotide of SEQ ID NO: 52. The DNA encoding fuzeon peptide thus obtained was amplified with PCR. The PCR was conducted under the condition of 30 cycles of amplification for 20 sec at 60 ^°C (annealing); and 20 sec at 68 ^°C (extension).

The vector pET22b was digested with Ndel and BamHI to delete signal sequences. The DNAs of the immunoglobulin fragment and the fuzeon peptides obtained by PCR were digested with restriction enzymes of Ndel and BamHI, respectively, and the resulting DNAs were cloned into vector pET22b, which was previously digested with Ndel and BamHI, using T4 DNA ligase. The expression vector thus obtained was designated immunoglobulin fragment-fuzeon (pFFuzeon) and the cloning procedure thereof is shown in Fig. 4.

2) Construction of immunoglobulin fragment-fuzeon-fuzeon expression vector

An expression vector for expressing a fusion protein composed of immunoglobulin fragment-fuzeon fusion protein and fuzeon peptides was constructed. The expression vector comprises a DNA encoding the immunoglobulin fragment-fuzeon fusion protein prepared in 1) above. 3' terminal of the immunoglobulin fragment-fuzeon fusion protein-coding sequence is ligated with 5' terminal of fuzeon peptide-coding sequence by in-frame fusion. In order to construct expression vectors for the fusion protein, a DNA encoding the immunoglobulin fragment-fuzeon fusion protein was synthesized by

PCR (30 cycles of amplification for 30 sec at 60 ^°C (annealing); and 60 sec at

68⁰C (extension)) using the oligonucleotides of SEQ ID NOs: 49 and 53, and the

DNA encoding the immunoglobulin fragment-fuzeon expression vector prepared in 1) above as a template DNA. At this time, the forward oligonucleotide of

SEQ ID NO: 49 was synthesized in the same manner as described in 1) above, and the reverse oligonucleotide of SEQ ID NO: 53 was synthesized to include only "p" (meaning phosphate) without inserting a restriction enzyme site at 5' terminal for in-frame fusion with fuzeon pepetides.

Fuzeon peptides were synthesized using SEQ ID NOs: 51 and 52 in the same manner as described in 1) above. Vector pET22b was digested with Ndel and BamHI to delete signal sequences. The DNAs of the immunoglobulin fragment and the fuzeon peptide obtained by PCR were digested with restriction enzymes of Ndel and BamHI, respectively, and the resulting DNAs were cloned into vector pET22b, which was previously digested with Ndel and BamHI, using

T4 DNA ligase. The expression vector thus obtained was designated immunoglobulin fragment-fuzeon-fuzeon (pFFuzeon-Fuzeon) and the cloning procedure thereof is shown in Fig. 4.

3) Construction of immunoglobulin fragment-C34 fuzeon expression vector

An expression vector for expressing a fusion protein of immunoglobulin fragment and C34 fuzeon peptide was constructed. The expression vector comprises a DNA encoding the human immunoglobulin Fc fragment of SEQ ID NO: 1 consisting of IgG4 hinge region, and CH2 and CH3 domains of the constant region of heavy chain of IgG4. 3' terminal of the immunoglobulin fragment-coding sequence is ligated with 5' terminal of C34 fuzeon peptide- coding sequence of SEQ ID: 64 by in- frame fusion.

In order to construct the expression vector for fusion protein, the DNA encoding the human immunoglobulin fragment was synthesized based on the blood cDNA library of Clontech laboratories Inc. A forward oligonucleotide of SEQ ID NO: 49 was synthesized in the same manner described in 1) above, and a reverse oligonucleotide of SEQ ID NO: 54 was synthesized to include some amino terminal sequences of C34 fuzeon peptides and "p" (meaning phosphate), without inserting a restriction enzyme site at 5' terminal site for in-frame fusion with C34 fuzeon peptide-coding sequence. C34 fuzeon peptide was synthesized by PCR using the DNA encoding immunoglobulin fragment-UB -fuzeon prepared in 1-3 above as a template DNA. For in-frame fusion with the immunoglobulin fragment, only "p" (meaning phosphate) was inserted into the 5' terminal of forward oligonucleotide of SEQ ID NO: 55 and Sail restriction enzyme recognition site was inserted into 3' terminal of reverse oligonucleotide of SEQ ID NO: 56. The DNA encoding C34 fuzeon peptide thus obtained was amplified with PCR using the oligopeptides. The PCR was conducted under the condition of 30 cycles of amplification for 30 sec at 60 °C (annealing); and 60 sec at 68 °C (extension).

The vector pET22b was digested with Ndel and Sail to delete signal sequences. The DNAs of the immunoglobulin fragment and the C34 fuzeon peptide obtained by PCR were digested with restriction enzymes of Ndel and Sail, respectively, and the resulting DNAs were cloned into vector pET22b using T4 DNA ligase. The expression vector thus obtained was designated immunoglobulin fragment-C34fuzeon (pFC34Fuzeon) and the cloning procedure thereof is shown in Fig. 4.

Example 2: Expression of fusion protein/peptide

BL21DE3 (E. coli B F-dcm ompT hsdS (rB-mB-) gal λ (DE3); Stratagene) was transformed with the expression vectors prepared in Example 1 according to the manufacture's protocols. After harvesting the transformed colonies, the colonies were seeded in a 2X Luria broth (LB) containing 50 /zg/ml of ampicillin and cultured at 37 ^°C for 15 hours. 1 ml of 2X LB medium containing the cultured medium and 30% glycerol in a ratio of 1 :1 (v/v) was added to a cryo-tube and stored at -140⁰C , to use as a cell stock. The resulting cells (transformants) designated as described in Table 1. Also, the transforaiants E. coli HMFOOl and HMF003, which are transformed with the expression vector pFUBPTHl-84 and pFUBIGF, respectively, were deposited at Korea Culture Center of Microorganism (KCCM) on 15^th January, 2009 under the accession numbers of KCCM 10980P and KCCM 1098 IP, respectively. <Table 1>

One vial of each of transformed cells was seeded in a 500 ml of 2X LB, and cultured at 37 ^°C for 14 to 16 hours with shaking. When the O.D. value at 600 nm of the culture reached 5.0, the culture was seeded into a 1.7 L of feπnentation medium containing 2% trypton, 1% yeast extract and 1% NaCl for initial batch culture using 5 L fermentor (MDL-8C, B. E. MARUBISHI, Japan). The fermentor was maintained at a temperature of 37^°C , aeration rate of 20 sL/min (lwm), and stirring speed of 500 rpm, and pH was adjusted to 6.70 using 30% ammonia water.

When the shortage of energy sources was caused by the growth of the microorganisms, the culture was subjected to a fed-batch culture while adding a feeding solution comprising 2OX yeast extract and 35% glucose to the culture. The growth of the microorganisms was monitored by determining the absorbance, and when the O.D. value at 600 nm of the culture reached 70, 100 uM of IPTG (total concentration) was added to the culture. The fermentation was further carried out for 23 to 25 hours. The fermented culture was centrifuged to obtain precipitated cells, which were then stored at -80 °C . The fermented recombinant fusion proteins showed about 5 g/L of expression level (at least 30% (w/w) of the total protein). Example 3: Recovery and refolding of recombinant fusion protein/peptide

In order to convert the recombinant fusion protein/peptide to a water- soluble form, the cells obtained in Example 2 were disrupted and the protein/peptide was refolded.

15 g of the cell pellets (wet weight) obtained in Example 2 was suspended in a 500 ml of dissolution buffer containing 50 mM Tris-HCl (pH 9.0), ImM EDTA (pH 8.0), 0.2M NaCl and 0.5% Triton X-100. The cells were disrupted using a microfluidizer process or M-110EH (AC Technology Corp., Model M1475C) at 15,000 psi twice. The disrupted cells were centrifuged at 12,000 XG at 40 ^°C for 30 minutes. The obtained supernatant was discarded, and the remainder was resuspended in 10 ml of Tris-HCl (pH 9.0) or distilled water and centrifuged at 12,000 XG at 40 "C for 30 minutes. The obtained supernatant was resuspended in a 200 ml dissolution buffer containing 8M urea and 50 mM of Tris-HCl (pH 8.0), and stirred at a room temperature for 2 hours. The resulting solution was centrifuged at 12,000 XG at 40 ^°C for 30 minutes to obtain a supernatant, and 1 mM of cystein was added to the supernatant with stirring for 30 minutes. And then, a refolding buffer containing 2 M urea, 0.75 M arginine, 0.5 mM cystein and 50 mM of Tris-HCl (pH 8.5) was added thereto, and the proteins in the reaction mixture were subjected to refolding for 12 to 16 hours.

Example 4: Purification of fusion protein/peptide

4-1) Protein A affinity column chromatography

To isolate a target fusion protein or peptide from the refolded sample obtained in Example 3, protein A affinity column chromatography (HiTrap rprotein A HP, Amersham Bioscience AB) was performed. Specifically, the refolded sample obtained in Example 3 was subjected to a desalting column to remove salts and the buffer was changed to 10 mM Tris-HCl (pH 8.0). The resulting sample was loaded on a protein A affinity column for binding the fusion protein/peptide in the sample to the immunoglobulin fragment (Carrier A) using its affinity, and eluted with 0.1M citric acid (pH 3.0) to obtain a fusion protein/peptide fraction. Tris-HCl buffer in an amount of 1/10 (v/v) of the obtained fraction was added thereto, to maintain the pH in a range of 7.0 to 8.0.

4-2) Q HP column chromatography

Anion exchange column chromatography (Amersham Bioscience AB) was conducted using the fraction obtained in Example 4-1. The fraction was subjected to a desalting column to remove salts, loaded on a Q HP column equilibrated with buffer A (10 mM Tris-HCl (pH 8.0)), and eluted with 5CV of buffer B (10 mM Tris-HCl (pH8.0) + IM NaCl) under a linear concentration gradient of 0 to 100%, to obtain a fusion protein/peptide fraction. The elution section was 5-20 ms/cm.

Example 5: Isolation of a target peptide

5-1) Cleavage of peptide using rEnterokinase

The fusion protein/peptide purified by protein A (HiTrap rprotein A HP; Amersham Bioscience AB) and Q HP column chromatography in Example 4 was concentrated to 1 mg/ml using vivaspin 20 (Sartorious stedim biotech). A mixture of 1OX rEK cleavage buffer and 10 unit rEK (Novagen) was added thereto, and the mixture was subjected to a reaction at a temperature of 20 ^°C for 16 hours. The yield of the peptide cleaved by rEK was determined by 15% criterion SDS-PAGE (bio-rad).

5-2) Cleavage of peptide using H6-UBP (ubiquitin hydrolase)

The fusion protein/peptide purified by protein A (HiTrap rprotein A HP; Amersham Bioscience AB) and Q HP column chromatography in Example 4 was concentrated to 1 mg/ml using vivaspin 20 (Sartorious stedim biotech). H6-UBP

(Advanced protein technologies corp.) was added in the ratio of 50: 1-100: 1 (substrate : H6-UBP) thereto, and the mixture was subjected to a reaction at a temperature of 30 "C for 2 to 3 hours. The yield of the peptide cleaved by H6- UBP was determined by 15% criterion SDS-PAGE (bio-rad). The results of SDS-PAGE carried out using the samples containing the fusion protein of immunoglobulin fragment and glucagon peptide, which were obtained each step of the purification process, are shown in Fig. 5.

In Fig. 5, lane 1 represents the fusion protein after refolding; lane 2, the fusion protein partially purified by Q column chromatography (before ubiquitin hydrolysis); lanes 3 to 6, hydrolysis products obtained by employing various concentrations of the fusion proteins and ubiquitin (lane 3 (2 mg of fusion protein : 80_/zg of ubiquitin); lane 4 (2 mg of fusion protein : 40_/zg of ubiquitin); lane 5 (4 mg of fusion protein : 80 μg of ubiquitin); and lane 6 (6 mg of fusion protein : 80^g of ubiquitin). As shown in Fig. 5, glucagon peptide was clearly separated from the fusion protein by H6-UBP (ubiquitin hydrolase).

5-3) SEC column chromatography

To isolate pure target peptides, SEC column chromatography was performed. The reaction mixtures prepared in 5-1 and 5-2 were respectively loaded on a sephadex 75 column previously equilibrated with a buffer of 10 mM Tris-Cl (pH8.0) + 100 mM NaCl at a flow rate of 2 ni/min. Then, the reaction mixture was eluted with the same buffer at a flow rate of 1 mi/min, to obtain pure target protein or peptide fractions. The SDS-PAGE results of glucagon, IGF-I, PTH 1-84 and fuzeon peptides are shown in Figs. 6A to 6D, and yields of the protein/peptide are represented in Table 2.

The results show that the present method makes it possible to attain a high level of expression of the target protein or peptide in a microbial host cell.

Example 6: Analysis of amino terminal sequence of peptides

In order to confirm whether the amino terminal sequences of the isolated peptides are identical with the original sequence of the peptides, sequencing analysis of amino terminal region was performed. As shown in Table 3, the isolated peptides had the same amino terminal sequence as that of the original sequence.

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

To. Hanmi Pharm. Co. Ltd

893-5, Hajeo-li, Paltan-myun, RECEIPT IN THE CASE OF AN ORIGINAL issued pursuant to Rule 7.1 by the Hwasung-gun, Kyunggi-do, INTERNATIONAL DEPOSITARY AUTHORITY KOREA identified at the bottom of this page

L

¹ Where Rule 6.4(d) applies, such date is the date on which the status of interfeSnaWepesftary was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authority. Form BP/4 Sole page BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

To. Hanmi Pharm. Co. Ltd

893-5, Hajeo-li, Paltan-myun, RECEIPT IN THE CASE OF AN ORIGINAL Hwasung-gun, Kyunggi-do, issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY KOREA identified at the bottom of this page

L

I . IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR : INTERNATIONAL DEPOSITARY AUTHORITY: Escherichia coli HMF003 KCCM10981P

H. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by:

D a scientific description

D a proposed taxonomic designation

(Mark with a cross where applicable)

EI. RECEIPT AND ACCEPTANCE

This International Depositary Authouity accepts the microorganism identified under I above, which was received by it on January. 08. 2009. (date of the original deposit)¹

IV. INTERNATIONAL DEPOSITARY AUTHORITY

Name : Korean Culture Center of Microorganisms Signature(s) of person(s) having the power to represent the International Depositary

Address : 361-221, Yurim B/D

Authority or of authori W! Hongje-1-dong, Seodaemun-gu SEOUL 120-091 Date: January. 08. 2009 ψm Republic of Korea ml W

¹ Where Rule 6.4(d) applies, such date is the date on which the status of in&iHBtwiiώ'βQjϋϊitary authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authority. Form BP/4 Sole page

Claims

WHAT IS CLAIMED IS:

1. A method for producing a physiologically active protein or peptide comprising the steps of: a) introducing a DNA coding for a fusion protein composed of an immunoglobulin fragment and a physiologically active protein or peptide into a cell, and culturing the cell; and b) isolating the fusion protein from the resulting cell culture and separating the physiologically active protein or peptide from the fusion protein.

2. The method of claim 1, wherein the fusion protein further comprises an enzyme-specific cleavage sequence disposed between the physiologically active protein or peptide and immunoglobulin fragment.

3. The method of claim 2, the enzyme-specific cleavage sequence is recognized by a protein selected from the group consisting of trypsin, thrombin, TEV protease, PreScission protease, enterokinase, and ubiqutin hydrolase.

4. The method of claim 1, wherein the immunoglobulin fragment is selected from the group consisting of the constant region of IgG₅ IgA, IgE, IgM, or IgD of human, mouse, pig, rabbit, or rat originated, and a combination or a hybrid thereof.

5. The method of claim 4, wherein the immunoglobulin fragment is selected from the group consisting of the constant region of IgGl, IgG2, IgG3, or IgG4, and a combination or a hybrid thereof.

6. The method of claim 5, wherein the immunoglobulin fragment is the constant region of IgG4.

7. The method of claim 1, wherein the physiologically active protein or peptide is selected from the group consisting of blood factor, digestive hormone, adrenocorticotropic hormone, thyroid hormone, intestinal hormone, cytokine, enzyme, growth factor, neuropeptide, hypophyseotropic hormone, hypophysiotropic hormone, anti-viral peptide, and and a non-native peptide derivative thereof retaining physiologically active property.

8. The method of claim 1, wherein the physiologically active protein or peptide is selected from the group consisting of erythropoietin, GM-CSF (granulocyte macrophage-colony stimulating factor), amylin, glucagon, insulin, somatostatin, PYY (peptide YY), NPY (neuropeptide Y), angiotensin, bradykinin, calcitonin, corticotropin, eledoisin, gastrin, leptin, oxytocin, vasopressin, LH (luteinizing hormone), prolactin, FSH (follicle stimulating hormone), PTH (parathyroid hormone), secretin, sermorelin, hGH (human growth hormone), growth hormone-releasing peptide, G-CSFs (granulocyte colony stimulating factor), interferons, interleukins, prolactin-releasing peptide, orexin, thyroid- releasing peptide, cholecystokinin, gastrin-inhibiting peptide, calmodulin, gastrin- releasing peptide, motilin, vasoactive intestinal peptide, ANP(atrial natriuretic peptide), BNP (barin natriuretic peptide), CNP (C-type natriuretic peptide), neurokinin A, neuromedin, renin, endothelin, sarafotoxin peptide, carsomorphin peptide, dermorphin, dynorphin, endorphin, enkepalin, T cell factor, tumor necrosis factor, tumor necrosis factor receptor, urokinase receptor, tumor inhibitory factor, collagenase inhibitor, thymopoietin, thymulin, thymopentin, tymosin, thymic humoral factor, adrenomodullin, allatostatin, amyloid beta- protein fragment, antimicrobial peptide, antioxidant peptide, bombesin, osteocalcin, CART peptide, E-selectin, ICAM-I, VCAM-I, leucokine, kringle-5, laminin, inhibin, galanin, fibronectin, pancreastatin, and fuzeon.

9. A fusion protein comprising an immunoglobulin fragment and a physiologically active protein or peptide, having the amino acid sequence of SEQ ID NO: 3.

10. A DNA encoding the fusion protein of claim 9.

11. An expression vector comprising the DNA of claim 10.

12. A microorganism transformed with the expression vector of claim 11.

13. The microorganism of claim 12, which is selected from the group consisting of E. coli HMFOOl (Accession NO: KCCM 10980P), HMF002, HMF003 (Accession NO: KCCM 10981P)₅ HMF004, HMF005, HMF006, HMF007, HMF008, HMF009, HMFOOlO, HMFOOIl, and HMF0012.

14. A method for mass-producing a physiologically active protein or peptide by using an immunoglobulin fragment as a fusion partner.