WO2004007740A1

WO2004007740A1 - Biological method for producing t-20 peptide

Info

Publication number: WO2004007740A1
Application number: PCT/KR2003/001384
Authority: WO
Inventors: Chung Kyung Kang; Jeong Hyun Kim
Original assignee: Chung Kyung Kang; Jeong Hyun Kim
Priority date: 2002-07-12
Filing date: 2003-07-12
Publication date: 2004-01-22
Also published as: KR100451432B1; AU2003245166A1; KR20040006539A

Abstract

The present invention relates to a biological method of producing T-20 or a T-20-like peptide, which comprises a) joining a polynucleotide coding a signal sequence or an insoluble leading sequence to a polynucleotide coding T-20 or a T-20-like peptide to prepare a gene construct coding a fusion peptide; b) inserting the gene construct into a vector to obtain an expression vector; c) transforming a host cell with the expression vector; d) cultivating the transformed cell to express the fusion peptide; and e) removing the signal sequence or the insoluble leading sequence from the fusion peptide to recover T-20 or the T-20-like peptide.

Description

BIOLOGICAL METHOD FOR PRODUCING T-20 PEPTIDE

Field of the Invention

The present invention relates to a biological process for producing T-20 or T-20-like peptides.

Background of the Invention

T-20, a peptide corresponding to amino acids 638 to 673 of

gp41 protein, is known to inhibit HIV from infecting CD-4⁺ cells, as disclosed in US Patent Nos. 5,464,933 and 5,656,480.

A chemical method of synthesizing T-20 peptide is described in US Patent Nos. 6,015,881 and 6,281,331, but such methods which are composed of the steps of repeatedly adding an amino acid to extending peptide chain and/or combing of the short peptides to complete the whole peptide are time- consuming and expensive for mass production of the peptide.

Preferred are biological methods for economical mass-producing relative long peptides, which are cost-effective and environmentally friendly, and such methods are disclosed in Korean Patent Nos. 263583 and 319529, and US Patent No. 6,183,992, etc.

In spite of advantage of the biological method for mass production of long peptide over chemical method, the biological method has obstacles to be overcome; said obstacles may be instability of a peptide in expression host, and the difficulty of economically and safely recovering an interest peptide from a fusion partner attached for improving its stability and purifying the peptide in a pure from.

The present inventors have therefore endeavored to overcome these difficulties of biological production, to provide a biological method for economically producing T-20 or a T-20-like peptide in a host cell in the form of a fusion peptide and recovering the T-20 or T-20-like peptide therefrom.

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:

FIGs. 1A and IB: the construct T20.1 and the construct T20.2, respectively;

FIGs. 2A and 2B: the construct G3T20 and the expression vectors thereof pET-G3T20, pHexl-G3T20 and pMexl-G3T20;

FIGs. 3 A and 3B: the construct G8T20 and the expression vectors thereof G8T20 pET-G8T20, pHexl-G8T20 and pMexl-G8T20; FIGs. 4A and 4B: the construct ompA sig and the construct ompT sig, respectively;

FIG. 5: the results of loading E.coli BE21(DE3) cell extracts transformed with pET-20.2, pET-G3T20, pET-G8T20, pET-ompAT20 and pET-ompTT20, respectively, in 16% tris-tricine polyacrylamide gel; FIG. 6: the results of loading E.coli W3110 cell extracts transformed with pHexl and pMexl, in 16% tris-tricine polyacrylamide gel;

FIG. 7: the process for lepB gene cloning;

FIGs. 8A and 8B: the cleavage of fusion peptides G8T20T by LepB, respectively; FIG 9: the construct G8.1 ;

FIG. 10: the results of loading E.coli BE21(DE3) cell extracts transformed with pET-G8.1T20.3, pET-G8.1T20.4, pET-G8.1T20.5 and pET- G8.1T20.6, respectively, in 16% tris-tricine polyacrylamide gel;

FIG. 11: the results of loading E.coli BE21(DE3) cell extracts transformed with pET-lepB4T20, pET-lepB5T20 and pET-lepB6T20, respectively, in 16% tris-tricine polyacrylamide gel(In Lanes T, S and P, total cell extracts, supernatants and precipitants of centrifuged cell crude extract were loaded, respectively).

Summary of the Invention

It is a primary object of the present invention to provide a biological method of producing fusion peptides containing T-20 or a T-20-like peptide joined with a signal sequence or an insoluble leading sequence. It is another object of the present invention to provide a method of recovering T-20 or the T-20-like peptide by cleaving said fusion peptide employing a peptidase.

Detailed Description of the Invention

The present invention also relates to a polynucleotide comprising a DNA sequence coding T-20 peptide, which is preferably the polynucleotide of SEQ ID NO: l.

T-20 peptide corresponds to amino acids 638 to 673 of HIV- I_LAI gp41 and the term "T-20 peptide" as used herein refers to native T-20 peptide without modification at its N- or C-terminus.

The term "T-20-like peptide" as used herein refers to a peptide having at least 70% homology with the amino acid sequence of T-20 peptide.

The term "a signal sequence" refers to an amino acid sequence which is joined with a protein or a peptide to form a fusion protein or peptide, thereby leading the protein or peptide to be integrated into the cytoplasmic or outer membrane, or secreted to the periplasm or culture medium.

The term "an insoluble leading sequence" refers to an amino acid sequence which is joined with a protein or peptide to form an insoluble inclusion body.

In step a) of the above method, a polynucleotide coding T-20 or a T-20- like peptide may be prepared by a biological or chemical method. Preferably, the polynucleotide may be chemically synthesized in consideration of the preferred codon usage of a host microorganism according to such a conventional method as PCR. The polynucleotide may comprise restriction sites for joining a fusion partner and cloning with a vector.

In order to produce T-20 or a T-20-like peptide in a microorganism on a large scale, it is necessary to make the peptide to be accumulated within the cell, get integrated into the cell membrane, or secreted to a culture media in an effective and stable fashion. For this purpose, T-20 or the T-20-like peptide may be designed to be expressed with a fusion partner such as a signal sequence or an insoluble leading sequence.

A gene coding the signal sequence or the insoluble leading sequence is joined to a gene coding T-20 or the T-20-like peptide to obtain a gene construct of the present invention.

Exemplary signal sequences of the present invention include signal sequences of bacteria or filamentous phage of Inoviridae, preferably, E.coli phage

Ml 3 gene III signal sequence (G3), E.coli phage Ml 3 gene VIII signal sequence

(G8), E.coli ompT signal sequence and E.coli ompA signal sequence, and, more preferably, E.coli phage M13 gene III signal sequence (G3) and E.coli phage M13 gene VIII signal sequence (G8).

Preferably used as the insoluble leading sequence of the present invention is an N-terminal sequence of matured LepB of E.coli, more preferably, the sequence of amino acids 76 to 130 of LepB, and most preferably, the sequences of amino acids 79 to 91, 79 to 110, and 76 to 129 of LepB.

The fusion peptide having a fused signal sequence is integrated into the cell membrane where a protease or a peptidase is not easily accessible, and, the fusion peptide having a fused insoluble leading sequence forms an insoluble inclusion body which is not degraded by a protease or peptidase, thereby allowing facile expression of T-20 or a T-20-like peptide in the form of a fusion peptide.

The gene construct coding the fusion peptide of the present invention may be produced according to known methods in the art, e.g., PCR or ligation by ligase. The construct may be a multimer comprising repeating units.

In step b) of the inventive method, the gene construct of the present invention is inserted into such a vector as a plasmid, virus or other vehicle.

The gene construct of the present invention may comprise a promoter, e.g., tac promoter, trc promoter, trp promoter, T7 gene 10 promoter, P_L promoter, other induced promoter or constitutive promoter, depending on the kind of microorganism used. Further, as a selective marker, the gene construct may comprise an antibiotic resistance gene (e.g., ampicillin, kanamycin, tetracycline or chloramphenicol) or a gene for compensating the nutritional requirements of a host cell.

In step c) of the inventive method, a host cell such as E.coli, Bacillus, streptomyces or yeast may be used. As an E.coli strain, BL21(DE3), BLR(DE3), B834(DE3), AS494(DE3), JM109(DE3), HMS174(DE3), UT400(DE3), UT5600(DE3), W3110, JM109, DH1 or TGI may be employed .

In step d) of the inventive method, one of the following culture media may be employed with or without added growth factors:

LB medium (Bacto-trypton lOg/1, yeast extracts 5g/l, NaCl lOg/1); M9 medium (Na₂PO₄.7H₂O 12.8g/l, KH₂P0₄ 3.0g/l, NaCl 0.5g/l, NH₄C1 lg/1, glucose 4g/l, MgSO₄2mM, CaCl₂ O.lmM);

M9CA medium (M9 medium + 0.2% casaminoic acid); R9 medium (Reisenberg medium; KH₂P0₄ 13.3g/l, (NH₄)₂HP0₄ 4.0g/l, citric acid 0.17g/l, MgSO .7H₂0 0.22g/l, glucose 20g/l, trace element solution lOml/1); and

Trace element solution (ferric citrate 7.3g/l, CoCl₂.6H₂0 0.5g/l, MnCl₂.4H₂0 3.2g/l, CuCl₂.2H₂0 0.3g/l, H₃B0₃ 0.7g/l, NaMo0₄.2H₂0 1.68g/l, thiamine HCI 0.5g/l, EDTA lg/1).

In step e) of the inventive method, if the fusion peptide is integrated into the cell membrane, the cells are disrupted, and then, signal peptidase, preferably

LepB, is added thereto so as to remove the signal peptidase. If the fusion peptide is secreted to the periplasmic space or medium, the signal peptide is spontaneously removed during the secretion process.

Further, a DNA sequence coding an amino acid sequence that can be cleaved by a chemical or protease, may be inserted between the fusion partner and the T-20 or T-20-like peptide.

The present invention is further described in the following Examples which are given only for the purpose of illustration, and are not intended to limit the scope of the invention.

Example 1: Preparation of the construct T20.1 coding T-20 peptide

A construct of SEQ ID NO: 1(T20.1 construct) was prepared as follows: 5μg of whole-F(SEQ ID NO: 2) and 5μg of whole-R(SEQ ID NO: 3) were dissolved in 50 μl of STE buffer(100 mM NaCl, 10 mM Tris-HCl (pH 8.0), lmM EDTA), denaturated at 95°C for 5 min., slowly cooled to room temperature to be annealed, and added thereto were 20 μl of 10X Klenow reaction buffer solution(500 mM Tris-HCl(pH 7.2), lOOmM MgS0₄, lmM DTT), 8 μl of 10 mM dNTP, 3 μl of DNA polymerase I large fragment (Klenow) (lOU/μl) and 119 μl of H₂0. Then, the mixture was reacted at 37°C for 3 hr to obtain T20.1 construct (See FIG. 1A).

The reaction mixture was heated at 65°C for 15min. to inactivate the enzyme, concentrated to 50 μl by ethanol precipitation, and then, treated with Kpnl/Hindlll. The resulting mixture was electrophoresed in 2% agarose gel, and a 125bp ds DNA was eluted, purified using Qiagen elution kit(QIAGEN, Inc. USA) and the purified DNA was cloned into pUC19.

Example 2: Preparation of the construct T20.2 coding T-20 peptide

A construct of SEQ ID NO: 4(T20.2 construct) coding the T-20 peptide was designed to contain the initiation codon ATG in front of T-20 coding region so as to be expressed without a fusion partner.

In order to synthesize the T20.2 construct, a PCR was performed employing T20F2 of SEQ ID NO: 5 and whole-R as a primer pair, and the cloned T20.1 prepared in Example 1 as a template to obtain T20.2 construct (See FIG. IB) under the following conditions:

<Reaction solution> Template DNA (50 ng/ μl) 1 μl

10X Taq reaction buffer 5 μl dNTP (lOmM) 1 μl

Primer T20 F2 (10 pmol/ μl) 1 μl

Primer Whole R (10 pmol/ μl) 1 μl Taq polymerase 1 μl

H₂0 40 μl

<Reaction condition> initial denaturation : 95 °C 5min. 1 cycle denaturation: 95°C 30 sec. annealing : 55.8°C 30 sec. elongation: 72°C 30 sec. 30 cycle final extension : 72 °C 5 min. 1 cycle

The PCR product was treated with phenol/chloroform to inactivate Taq

DNA polymerase, concentrated to 20 μl by ethanol precipitation, and then digested with Ndel/Hindlll. The resulting mixture was electrophoresed in 2% agarose gel, and a 116bp DNA fragment was eluted and purified using Qiagen elution kit(QIAGEN, Inc. USA). Plasmid pUC19 was digested with Ndel/Hindlll and the 116bp DNA fragment was inserted thereinto to obtain plasmid pUC-T20.2.

Plasmid pUC-T20.2 was again digested with Ndel/Hindlll to obtain a 116bp DNA fragment (the construct T20.2). Plasmid pET24a (Novagen, USA) was treated with Ndel/Hindlϊ , and the T20.2 construct was inserted thereinto to obtain plasmid pET-T20.2.

Example 3: Preparation of G3T20 construct coding a fusion peptide, T-20 peptide joined with signal sequence of E. coli phage M13 gene III (G3)

In order to prepare G3T20 construct, a PCR was performed employing the cloned T20.1 construct prepared in Example 1 as a template DNA, and

G3T20F1 of SEQ ID NO: 7 and whole-R as a primer pair to obtain a G3T20 construct of SEQ ID NO: 6 (See FIG. 2A). The PCR conditions were the same as described in Example 2 except the annealing temperature was 56 ^°C . The PCR product was treated with phenol/chloroform, concentrated to

20 μl by ethanol precipitation, and digested with Ndel/Hindlll. The resulting mixture was electrophoresed in 2% agarose gel, and a 167bp DNA fragment was eluted and purified using Qiagen elution kit(QIAGEN, Inc. USA).

Plasmid pET24a was digested with Ndel/Hindlll, the 167bp DAN fragment was inserted thereinto to obtain plasmid pET-G3T20, and then, plasmid pET-G3T20 was again digested with Ndel/Hindlll to obtain 167bp DNA fragment. Plasmids pHexl and pMexl were treated with Ndel/Hindlll, and the 167bp DNA fragment was inserted thereinto to obtain plasmids pHexl-G3T20 and pMexl-G3T20, respectively(See FIG. 2B).

Example 4: Preparation of G8T20 construct coding a fusion peptide, T-20 peptide joined with signal sequence of E. coli phage M13 gene VIII (G8)

In order to prepare a G8T20 construct, a PCR was performed employing the cloned T20.1 construct prepared in Example 1 as a template

DNA, and G8T20F1 of SEQ ID NO: 9 and whole-R as a primer pair to obtain a

G8T20 construct of SEQ ID NO: 8 (See FIG. 3A). The PCR conditions were the same as described in Example 2 except the annealing temperature was 56 °C .

The PCR product was treated with phenol/chloroform, concentrated to 20 μl by ethanol precipitation, and digested with Ndel/Hindlll. The resulting mixture was electrophoresed in 2% agarose gel, and a 182bp DNA fragment was eluted and purified using Qiagen elution kit(QIAGEN, Inc. USA).

Plasmid pET24a was digested with Ndel/Hindlll, the 182bp DNA fragment was inserted thereinto to obtain plasmid pET-G8T20, and then, plasmid pET-G8T20 was again digested with Ndel/Hindlll to obtain the 182bp DNA fragment. Plasmids pHexl and pMexl were treated with Ndel/Hindlll, and the 182bp DNA fragment was inserted thereinto to obtain plasmids pHexl-G8T20 and pMexl-G8T20, respectively (See FIG. 3B).

Example 5: Preparation of ompAT20 and ompTT20 constructs coding fusion peptides, T-20 peptides joined with ompA and ompT signal sequences, respectively

First, 5μg each of ompAl of SEQ ID NO: 12 and ompA2 of SEQ ID NO: 13 was dissolved in 50 μl of STE buffer (100 mM NaCl, 10 mM Tris-HCl (pH 8.0), lmM EDTA), denaturated at 95°C for 5 min., and then, slowly cooled to room temperature to be annealed to obtain a gene construct (ompA sig) coding the ompA signal sequence(See FIG. 4A). Likewise, a gene construct (ompT sig) coding the ompT signal sequence was prepared by the same method except for employing ompTl of SEQ ID NO: 14 and omρT2 of SEQ ID NO: 15 instead of ompAl and ompA2 (See FIG. 4B).

T20.1 construct prepared in Example 1 was digested with Accl/Hindlϊl to obtain a lllbp DNA fragment, ligated with 2 μg of ompA sig or orripT sig. The ligated product was precipitated by ethanol, treated with Ndel/HindUl, and the mixture was electrophoresed in 2% agarose gel. Then, 160bp and 173bp DNA fragments, which represents constructs ompAT20(SEQ ID NO: 16) and ompTT20(SEQ ID NO: 17), respectively, were each eluted from the gel and purified using Qiagen elution kit (QIAGEN, Inc. USA). Plasmid pET24a was treated with Ndel/Hindlll and the ompAT20 and ompTT20 constructs were inserted thereinto to obtain plasmids ET-ompAT20 and pET-ompTT20, respectively.

Example 6: Expression of T-20 peptide, and fusion peptides G3T20, G8T20, ompAT20 and ompTT20

E.coli BE21(DE3) was transformed with each of plasmids pET-T20.2, pET-G3T20, pET-G8T20, pET-ompAT20 and pET-ompTT20 prepared in Examples 2 to 5. The transformed cells were grew in LB medium containing 50 μg/ml of kanamycin for 12hr at 37^°C, and then, the seed culture was inoculated into a fresh medium at a ratio of 1/100 (v/v).

For inducing the expression of the respectively peptides, 2mM of IPTG or 2% lactose was added to the medium when the optical density(OD₆oo) of the medium reached 0.5, and the cells were cultivated further for 4hr. Then the cells were harvested from the medium by centrifugation at 6,000rpm, and electrophoresed in 16% tris-tricine polyacrylamide gel. The result is shown in FIG. 5.

The lanes for E.coli BE21(DE3) transformed with pET-G3T20, pET-

G8T20, ρET-omρAT20 and pET-omρTT20 showed the bands corresponding to the fusion peptides G3T20, G8T20, omρAT20 and omρTT20, respectively, while the lane for pET-T20.2 did not show the band corresponding to the peptide T-20(See FIG. 5).

To confirm the stable expression of the fusion peptides G3T20 and G8T20, E.coli W3110 was transformed with pHexl-G3T20, pMexl-G3T20, pHexl-G8T20 and pMexl-G8T20, respectively, according to the above method. Likewise, G3T20 and G8T20 were also expressed in a stable manner (FIG. 6).

Example 7: Removing fusion partner from the fusion peptide by LepB

(1) Cloning lepB gene coding signal peptidase I (LepB)

For cloning the LepB gene coding signal peptidase I, a PCR was performed using E.coli JM109 chromosome as a template DNA, and lepBF of

SEQ ID NO: 10 and lepBR of SEQ ID NO: 11 as a primer pair to obtain a PCR product comprising lepB gene. The PCR conditions were the same as described in Example 2.

The PCR product was treated with phenol/chloroform, concentrated to

20 μl by ethanol precipitation, and digested with Ndel/Hindlll and Hindlll/Pstl, respectively. The resulting mixture was electrophoresed in 2% agarose gel and then, 424bp and 562bp DNA fragments were isolated and purified using Qiagen elution kit(QIAGEN, Inc. USA). pET24a and ρUC119 were treated with Ndel/Hindlll and Hindlϊl/Pstl, respectively, and the 424bp and 562bp DNA fragments were inserted thereinto to obtain plasmids lepB400 and lepB593, respectively.

In order to clone the LepB gene comprising RBS (Ribosome Binding Site) of T7 gene 10 and assemble the whole LepB gene, plasmids lepB400 and lepB593 were digested with Xbal/Hindlϊl and Hindlll/Pstl, respectively, to obtain 462bp and 562bp DNA fragments. The DNA fragments were ligated each other and treated with Xbal/Pstl to obtain a 1024bp DNA fragment. The 1024bp DNA fragment was ligated with a Xbal/Pstl fragment (3.1kb) of plasmid pUC118 to obtain plasmid pUC118-lepB containing lepB gene under the control of lacZ promoter.

Further, the pUC118-lepB was partially digested with Xbal/Hindlϊl to obtain a 1032bp DNA fragment, which was ligated with Xbal/Hindlll fragment(2.3kb) of plasmid pMex2r to obtain plasmid pMex2r-lepB(See FIG. 7).

(2) Cleavage of fusion partner from peptides G8T20

(i) Cleavage of fusion partner from peptide G8T20 by crude LepB protein

E.coli BL21(DE3) cells transformed with pET-G8T20 were disrupted by sonication, and centrifuged at 30,000 rpm to obtain a membrane fraction. E.coli JM109 cells transformd with pUC118-lepB was treated according to the same method as above, to obtain a membrane fraction. The membrane fractions of the BL21(DE3) and JM109 cells were mixed at a ratio of l:l(v/v), reacted at 30°C/37°C for 30min/60min and then the extent of cleavage was analyzed by 16%> tricine gel electrophresis.

As a result, 40% of the T-20 peptide was recovered from the fusion peptide, judged by quantitation of T20 produced from scanning of the gel with densitometer (See FIG. 8 A).

(ii) Cleavage of fusion partner from peptide G8T20 by two vector system

E.coli BL21(DE3) was cotransformed with pMex2r-lepB and pET24a, respectively, disrupted by sonification, and centrifuged at 4,000 rpm to obtain a supernatant.

The supernatant was incubated at 30°C/37°C for 30min/60min, and then, the extent of cleavage was analyzed by 16%> tricine gel electrophresis.

The result showed that almost all of the T-20 peptide was freed by cleavage from the fusion peptide G8T20 (See FIG. 8B).

Example 8: Preparation of G3T20CNBr construct

In order to prepare a construct, G3T20CNBr construct of SEQ ID NO: 18, which codes the G3 signal sequence and T-20 peptide as well as CNBr- cleavable methionine located therebetween, a PCR was performed employing the T20.1 construct prepared in Example 1 as a template DNA and G3T20F2 of

SEQ ID NO: 19 and Whole-R as a primer pair. The PCR conditions were the same as described in Example 2. The PCR product was treated with .phenol/chloroform, concentrated to

20 μl by ethanol precipitation, and digested with Ndel/Hindlll. The resulting mixture was electrophoresed in 2% agarose gel, and the 177bp DNA fragment was isolated and purified by using Qiagen elution kit(QIAGEN, Inc. USA). pET24a was digested with Ndel/Hindlll, the 177bp DNA fragment was inserted thereinto to obtain pET-G3T20CNBr.

According to the method of Example 6, the fusion peptide G3T20CNBr was expressed in E.coli BL21(DE) transformed with pET-G3T20CNBr. The result showed that the amount of the expressed fusion peptide G3T20CNBr was similar to that of G3T20 of Example 6. As a result, at least 60% of the T-20 peptide was recovered from the fusion peptide G3T20CNBr.

Example 9: Preparation of the gene construct coding fusion peptides, T20- like peptides joined with G8.1 and expression thereof A gene construct coding a derivative of G8 signal sequence, G8.1 construct(SEQ ID NO: 20) was preρared(See FIG.9). G8.1 construct was designed to have nucleotides coding amino acids 1 to 18 of G8 signal sequence. However, G8.1 construct comprises CTG and GTA as codons corresponding to 16th amino acid (Leu) and 17th amino acid (val), instead of CTC and GTT of that coding G8 signal sequence, respectively, in order to introduce a restriction enzyme site (Kpnϊ) thereinto for joining with a T20-like peptide.

For this purpose, G8F1 construct (SEQ ID NO: 21) and G8R1 construct (SEQ ID NO: 22) were denaturated and annealed according to the method of Example 1.

Four gene constructs coding T20-like peptides, T20.3 (SEQ ID NO: 23), T20.4 (SEQ ID NO: 26), T20.5 (SEQ ID NO: 28) and T20.6 (SEQ ID NO: 30) constructs, were prepared by PCRs. Primer pairs and templates employed herein are listed in Table 1.

Table 1

The G8.1 construct was treated with Ndel/Kp l, and the DNA constructs coding T20-like peptides (T20.3, T29.4, T20.5 and T20.6 constructs) were digested with Kpnl/Hindlll, respectively. Then, the G8.1 construct, and each of the constructs coding T20-like peptides (T20.3, T29.4, T20.5 and T20.6 constructs) were ligated together, followed by isolating and purifying the ligated product using Qiagen elution kit (QIAGEN, Inc. USA). pET24a was digested with Ndel/Hindlll, each of the DNA fragments was inserted thereinto to obtain ρET-G8.1T20.3, pET-G8.1T20.4, pET- G8.1T20.5 andpET-G8.1T20.6.

According to the method of Example 6, pET-G8.1T20.3, pET- G8.1T20.4, ρET-G8.1T20.5 and ρET-G8.1T20.6 were each expressed in E.coli BL21(DE) transformed therewith. As a result, each of the fusion peptides containing T20-like peptides was observed to be expressed in BL21(DE3), respectively(See FIG. 10).

Example 10. Fusion with insoluble leading sequence lepB4, lepB5, lepB6 and expression thereof

Selected as gene constructs coding insoluble leading sequence of fused T-20 peptides were those coding lepB4, which corresponds to the amino acids 79 to 91 of LepB; lepB5, 79 to 110 thereof; and leρB6, 76 to 129 thereof.

The insoluble fusion partners were prepared by denaturation and annealing according to the method of Example 1, as shown in Table 2.

Table 2

T20.1 construct prepared in Example 1 was digested with AccllHindlll to obtain a lllbp DNA fragment, ligated with 2 μg of lepB4, lepB5 or lepB6 construct. The mixture was precipitated by ethanol, treated with Ndel/Hindlll, and the resulting mixture was electrophoresed in 2% agarose gel. Then, DNA fragments of 167, 224 and 281bρ which corresponded to leρB4T20, lepB5T20 and lepB6T20 constructs, respectively, were eluted and purified by using Qiagen elution kit (QIAGEN, Inc. USA), respectively. pET24a was treated with Ndel/Hindlll, and each of lepB4T20, lepB5T20 and lepB6T20 were inserted thereinto to obtain pET-lepB4T20, pET-lepB5T20 and pET-lepB6T20, respectively.

According to the method of Example 6, G8.1T20.3, G8.1T20.4, G8.1T20.5 and G8.1T20.6 constructs were expressed in E.coli BL21(DE) transformed therewith. Each of the fusion peptides G8.1T20.3, G8.1T20.4, G8.1T20.5 and G8.1T20.6 constructs was observed to be expressed in a stable fashion (See FIG. 11).

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.

Claims

What is claimed is :

1. A biological method of producing T-20 or a T-20-like peptide, which comprises a) joining a polynucleotide coding a signal sequence or an insoluble leading sequence to a polynucleotide coding T-20 or a T-20-like peptide to prepare a gene construct coding a fusion peptide; b) inserting the gene construct into a vector to obtain an expression vector; c) transforming a host cell with the expression vector; d) cultivating the fransformed cell to express the fusion peptide; and e) removing the signal sequence or the insoluble leading sequence from the fusion peptide to recover T-20 or the T-20-like peptide.

2. The method of claim 1, wherein step e) comprises cleaving the signal sequence or the insoluble leading sequence off the fusion peptide by the action of an enzyme selected from the group consisting of signal peptidase I (LepB), Factor Xa and enterokinase.

3. The method of claim 2, wherein the enzyme is signal peptidase l(LepB).

4. The method of claim 1, wherein step e) comprises cleaving the signal sequence or the insoluble leading sequence off the fusion peptide by the action of CNBr or hydroxylamine.

5. The method of claim 1, wherein the signal sequence is a signal sequence of filamentous phage (family : Inoviridae).

6. The method of claim 5, where the signal sequence of filamentous phage is selected from the group consisting of E. coli phage M13 gene III signal sequence, E. coli phage Ml 3 gene VIII signal sequence and derivatives thereof.

7. The method of claim 1, wherein the signal sequence is ompT signal sequence or ompA signal sequence.

8. The method of claim 1, wherein the insoluble leading sequence is an N- terminal fragment of LepB.

9. The polynucleotide coding T-20 which has the nucleotide sequence of SEQ ID NO: 1.