WO1985005619A1

WO1985005619A1 - Novel polypeptide and process for its preparation

Info

Publication number: WO1985005619A1
Application number: PCT/JP1984/000434
Authority: WO
Inventors: Masakazu Kikuchi; Tsutomu Kurokawa; Susumu Honda
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 1984-06-06
Filing date: 1984-09-11
Publication date: 1985-12-19
Also published as: WO1985005618A1; ZA853966B; JPS615096A; KR860000377A

Abstract

A novel polypeptide having a biological activity similar or superior to that of IFN-gamma and scarcely dimerizing or polymerizing, a transformant containing DNA capable of coding the novel polypeptide, and a process for preparing the novel polypeptide from a culture product of the transformant. The novel polypeptide-coding DNA can be prepared, for example, from known IFN-gamma gene (cDNA)-containing plasmid, pHITtrp1101 or pHITtrp1201. This DNA is bound to a vector and introduced into host to obtain a transformant. In purifying the polypeptide from the culture product, an antibody column is used. The polypeptide can be used as antiviral or antineoplastic agent.

Description

Handbook JLU オ '· h め. め.

TECHNICAL FIELD The present invention relates to a novel polypeptide useful as a pharmaceutical and the like, and a method for producing the same.

Background technology

r-type interferon (hereinafter sometimes abbreviated as IFN-II) is produced from immunocompetent cells in a situation where lymphocyte blast formation and lymphokine production occur. Also called interferon. Compared to IPNa and IFN / 3, Γ Γ N-r is said to have higher anti-hyperalgic activity and anti-higashikei activity, and is expected to be applied to floor applications. However, due to restrictions such as the necessity of producing new limp balls, efficient production yarns have not been isolated until now. '

However, recently, gene recombination technology has become widely used, and the complementary DNA (cDMA) of IF I;

The S3 sequence and its sequence have revealed the expected amino acid S sequence, and it has become possible to express cDMA and chemically synthesized genes using various hosts [Gray. f. et al.Nature, 295..503 (1982) Devos, R et al., Nucleic Acids.Rea, 10, 2487 (1982),

Tanak _f S et al., Nucleic Aoida Rea., U, 1707 (1983)]. Furthermore, the purification method using a monoclonal antibody enables large-scale production of I Fii-r (hereinafter sometimes abbreviated as riFir- _r ), which is a technology for genetic recombination. No. 0103898], and clinical application is also approaching. However, the rIFH-r obtained here is difficult to purify or formulate, because it forms dimers or multimers and is unstable. Therefore, advanced technology is essential for the purification and formulation. I'm wearing

The present inventors believe that the above-mentioned disadvantages of the known ri ρ Ν—r are based on the 2 僳 Cys present at the Νterminal, and that the biological activity is equivalent to or higher than that of r IPN-r Thus, the present invention succeeded in producing a new polypeptide which is less likely to cause dimerization and multi-Jt formation, and completed the present invention.

Disclosure of the invention

The present invention uses the formula

(K) H-X-Y-Asp Pro Tyr Val Lys Ql Ala Qlu Asn

Leu JB Ljs Tyr P e Asn Ala Qly His Ser As Yal Ala Asp Asn Qly Thr Leu Phe _¾ Leu Gly lie Leu Lys Asn Trp Lys Qlu Qlu Ser Asp Arg Lys lie Met Gin Ser Gin lie Y l Ser Ph.e Tyr Phe Lys -Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Val Qlu Thr lie Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Ljs Lya Lys Arg Asp Asp Phe Qlu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Yal Qln Arg Lys Ala lie His Glu Leu lie Gin Yal Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly— Z— OH (C) (I) Met or bond, Y is Cys-Gin, Gin, Gin or bond, Z is (N) Lya Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gin (C) Has 1 to 16 amino acids from its N-terminus in the chain Which represents an amino acid residue or an amino acid residue), an industrially advantageous production method thereof, and a transformant which can be used for the production thereof.

Regarding the polypeptide (I), X is preferably a bond. Y is preferably Cys-Oln.Qln or Gin, and! It is preferable that <Gln. Z is Lys or Ly — Arg— Lys— Arg— Ser— Gln-Met-Leu-Phe-Arg-Gly-Arg (H) or Lye-Arg-Lys-Arg— SβrGIn— Met— Leu -Phe—Arg-01 y—Arg—Arg—A1a-Ser-Gln (H) is preferred.

In particular, as a polypeptide (I), X is a bond, Ϊ is Cya-Gln or <Gln, and Z is Lye or ぺ butide (B). In the polypeptide (I), X is the bond O

Polypeptide (I) has, for example, ATG at the 5 terminus,

() Η- '-Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Aen Gly Thr Leu Phe Leu Qly lie Leu Lye Asn Trp Lys Glu Qlu Ser Asp Arg Lya lie Met Gin Ser Qln lie Yal Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lya Asp Asp Gin Ser lie Gin Lya Ser Val Glu Thr lie Lys Qlu Aep Met Asn Val Lye Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Qlu Lys Leu Thr Asn Tyr Ser Yal Thr Asp Leu Asn Val Gin Arg Lys Ala lie His Glu Leu lie Gin Val Met Ala

O PI Qlu Leu Ser Pro Ala Ala LJB Tr Gly Lye Arg Lye Arg Ser Gin Met Leu Phe "Arg Qly-Z-OH (C) (Ι ') where 式' is Cys ~ Gln, Gln or a bond Z represents (N) Lys Arg Lys Arg Ser Gin Met Leu Phe Arg GI7 Arg Arg Ala Ser Gi (C) A peptide or amino acid residue having 1 to 16 amino acids counted from its K-terminal in the peptide chain. Can be advantageously produced by culturing a transformant containing a region encoding the polypeptide represented by the following formula: and a DIfA having a translation termination codon.

In contrast to the above-mentioned DNA, the region encoding the polypeptide (I ′) may be any region having the nucleotide sequence encoding the above-described polypeptide (Γ).

(5 ^ Y ¹ -GAC CCA TAT QTA AAA GAA QCA OAA A AC CTT AAG AAA TAT TTT A AT GCA GGT CAT TCA- GAT GTA QCG GkT AAT GGA ACT CTT TTC TTA GQC ATT TTG AAG AAT TGG AAA GAG GAG AQ GAC AO A AAA ATA ATG CAG AGC CAA ATT GTC TCC TTT ΤλΟ TTC AAA CTT TTT AAA AAC TTT AAA GAT GAC CAG AGC ATC CAA A AO AGT GTQ GAG ACC ATC AAG GAA GAC ATG AAT GTC AAG TTT TTC AA AGC AAC AAA AAG AAA CGA GAT GAC TTC GAA AAG CTQ ACT AAT TAT TCG QTA ACT GAC TTG AAT GTC CAA CGC AAA GCA ATA CAT CAT GAA CTC ATC CAA GTQ ATG GCT GAA CTG TCG CCA GCA GCT AAA AC A GQG-Z ¹ (3 (DT) Ϊ ¹ indicates TGC CAG or CAG or a bond, and Zl indicates (50 A AG CGA AAA AGG AQT CAG ATG CTG TTT CGA GGT CGA AGA QCA TCC CAG (shows the S3 column from the 1 'to 16' codons counted from the 5 'end of the S3 column shown in ()). TGC CAG or CAG as divided I ¹ is, those having a ¾ group sequence represented by AAG CGA AAA AGQ AGT CAG ATO CTQ TTT CGA GGT CGA AGA GCA TCC CAG (V) as Z ¹ is preferred. Translation stop codons include T AA, TGA or TAG, with TAA being preferred.

The above DNA consists of a signal peptide between the terminal ATG and the DNA encoding polypeptide (Γ) ^. Lya—Tyr—Tlxr—Sβr-Tyr—11β—Len—Ala—Phβ — Ql n— L eu— Cys— 11 e— Val— Leu— G 1 y— S β r— L β u—G 1 y: DN A, for example, AAA TAT ACA AGT TAT ATC TTG GGT TTT CAG CTC TGC ATC QTT TTQ GGT TCT CTT GGC

May be provided.

The above-mentioned DHA preferably has a promoter upstream of ATG, and the promoter may be any promoter that is suitable for the host used for the production of the transformant.

For example, in Escherichia coli (eg, 294, W3110, DH1, etc.), the trp promoter, lac promoter, rec A promoter, λΡι_ promoter, lpp promoter *, etc. Baci- llua subtilis (eg, MI114), SP01 promoter *, SP02 promoter, I »nP promoter, etc. For yeast (Sacoha-roayces cerevisiae; eg, AH22) SV 0 for animal cells (eg, cell C 0 S-7, Chinese hamster cell CH 0, etc.) such as PH 0 5 mouth motor-, PG mouth motor, GAP promoter, ADH promoter, etc. Bromotor derived from 0 ? It is preferable to use a trp blower with E. coli as a host.

A DNA (brasmid) having an ATG at the 5 'end and a polypeptide (Γ) downstream thereof and a translation termination codon downstream thereof is a known IFN- produced by chemical synthesis or gene engineering. r-cDNA or IF-rD! TA derived from chromosome can be produced by processing.

In order to more specifically explain the above-mentioned production method, a known IFN-r gene

Brasmid containing (cDNA) pH IT ti 1101! : Refer to Reference Example 2 of EPC Publication No. 0103898 (leakage)] as a raw material, and describe a method for producing a D »A-containing brasmid having a region encoding holybebutide (Γ; Z is a peptide (H)). This is illustrated below.

By co-digesting the brasmid pHI Ttrpl 101 with a restriction enzyme such as Avail, Pstl, DNA encoding IFN-r lacking Cys-Tyr-Cys-Gin can be obtained from the Ιί end of IPli-r. Next, for example, a synthetic adapter containing ATG (start codon) and coding for Cye-Gin, a synthetic adapter containing start codon and coding for Gin, or a synthetic adapter containing start codon and containing a start codon are respectively subjected to the Trieste method [Crea, R et al. Proc. NatL Acad. ScL USA 75, 5765 (1978)] and ligate them to DNA encoding IPN-r lacking Cys-Tyr-Cys-Gin, respectively. Next obtained D N

A can be inserted downstream of an appropriate promoter to introduce it into an appropriate host, but if necessary]) An S D (Shine and Dalgarno) sequence may be inserted downstream of the promoter. The transformant of the present invention can be prepared using the expression plasmid obtained as described above in a manner known per se [Cohen S. N. et al .; Pro. HatL Acad. Sci. USA,

O PI 69.2110 (1972)].

Polypeptide (I) can be produced by culturing the above-described transgenic plant, producing and accumulating polypeptide (I) in the culture, and collecting this.

Examples of the medium include グルコース 9 medium containing glucose and casamino acid [

Miller, J, Experiments in Molecular Genetics, 31-433 (Cold Spring Horbor Laboratory, New York, 1972)]. Here you need it! For example, a drug such as 3-indry can be added in order to make the motor more efficient.

Culture is usually performed at 15 to 43 for 3 to 24 hours, and if necessary, aeration and / or agitation can be added.

When a recombinant having a cits repressor and an expression vector containing a PL promoter is used, cultivation is performed at a low temperature of about 30 to 36, and inactivation of the cits repressor is about 37-: Preferably at 42'C. In order to make the reoA promoter work more efficiently, that is, to decrease the recA gene expression suppression function, it is necessary to add an agent such as D-mitmycin C or nadixic acid as necessary and to irradiate with ultraviolet rays. Can be.

After culturing, the cells are collected by a known method and, for example, gently washed in a loose mouth liquid. After that, the cells are disrupted by, for example, treatment with a protein denaturant (@sonic wave) or treatment with lysozyme or the like, followed by centrifugation. Difficulty * Obtain the supernatant by a known method.

Polypeptide (I) in which Z is an amino acid residue under 15 J5t or polyamino acid residue has more amino acids than Z. (For example, Z is a peptide having all 16 amino acids described above). A transformant containing DNA having a region encoding a polypeptide (IT) is cultured, and the action of the protease in the transformant is determined. It can also be produced by purifying under conditions that are easily affected.

In order to isolate the polypeptide (I) from the above obtained above, a protein purification method generally known may be used. In particular, an antibody capable of binding to IFN- or polypeptide (I) can be advantageously purified using that antibody column. For example, HLys-Arg-Lys-Arg-Ser-Gln-1 et— Le — P he— Ar g— G 1 y— A rg— A rg— A 1 a— Ser— Gin—OH Monoclonal antibody antibody column [EPC Publication No. 0 103 Publication No. 898, Example 12 (Γ2-11 1 1 monoclonal antibody column), Example of filtration 18 (r3-11.1 monoclonal antibody), an antibody column prepared in the same manner as above) Gin-Asp--Pro-Tyr-Vl-Lys-Qlu-Ala-Glu-Asn-Leu-Lye-Lye-yr-Phe-Asn-Ala-Qly-OH Ram [Special Issue 58–2 151 168 No. 1 (January 15th, 1983)] Example of harm to the specification 11 (W! Ir 2-76.53 Monoclonal antibody) Column))) It is.

In order to purify the antibody with the above antibody column, for example, a polypeptide (I) -containing substance is dissolved in an S buffer near a neutral substance, for example, a phosphate buffer solution such as tris phosphate buffer solution, and the antibody activity is reduced. Adsorb to ram. Next, the column is washed with the same buffer solution, and the polypeptide (I) is eluted. The eluate used is a weakly acidic solution, such as vinegar solution, a solution containing polyethylene glycol, and more antibody than polypeptide (I)! A solution containing a peptide that easily binds, a highly concentrated solution, and a combination of these are used.

CDMPI Those which do not accelerate the decomposition of butide (I) are preferred.

The column eluate should be neutralized with a normal solution]). If necessary]? The purification operation using the above antibody power and antibody can be performed again.

In the case of the peptide (I), Y force 5Cys ~ G ln, (31n or, in the case of a bond, a mixture of a polypeptide in which X is a bond and that in which X is Met may be obtained.

In addition, when the 1-terminal amino acid in the repeptide (I) is (Un), it may be obtained as a mixture with the polypeptide (I) in which <Gln is present. If necessary, for example, after the above-mentioned purification operation, the polypeptide (I) in which the N-terminal amino acid is Gin can be obtained by heating or treating with diacid (eg, dilute acetic acid).

The polypeptide (I) solution obtained here is subjected to dialysis, if necessary! ? This can be lyophilized into powder. During freeze-drying, stabilizers such as Sovito-mannitol, dextroth, matose and glycerol * can be added.

Since the polypeptide (I) thus obtained has only one or no Cys, it can be obtained as a stable monomer which is less susceptible to dimerization or multimerization than IFN-r in the prior art. In this case, precipitation hardly occurs, and the biological activity of β-time * decrease is leveraged to be small, so that it can be advantageously used as pharmaceuticals.

Helsingborg peptide (I) of the present invention, Boripe Buchido having 1 0 ⁷ specific activity of more than in WE scan activity measurement by cytopathic effect inhibition test of vesicular stomatitis Wie scan for对human amnion-derived WISH cells (VSV) It can be purified to a known level, and it is known that Naturally derived I * 1 * 1 r type 2 IFN) C Salvin et aL, J

It can be used for the same purpose as that of National Cancer Ins titute,, 123 (1975)].

The polypeptide (I) of the present invention exhibits antiviral, antitumor, cell growth inhibitory and immunopotentiating effects. The polypeptide (I) of the present invention can be mixed with sterilized water, human blood albumin (HSA), physiological saline, and other known physiologically acceptable carriers, and can be parenterally or topically. «Can be given. For example, 100,000 to 1 image cut per adult, preferably 400000 to 400000 cut per day, by intravenous injection or intramuscular injection! ) Can be administered.

Formulations containing ^ Lipeptide (I) of the present invention may contain other physiologically acceptable active ingredients such as ¾, diluents, adjuvants, other carriers, buffers, binders, surfactants, and preservatives. May also be contained. Preparations for intraoral administration may be sterile aqueous solutions or suspensions in physiologically acceptable soot, or sterile powders (usually Boribabe) which can be diluted and used in physiologically acceptable diluents. (Obtained by freeze-drying the peptide (I) solution).

Further, the preparation containing the ribeptide (I) of the present invention comprises an active ingredient such as IFN-a, IFN- or IFK-r or interleukin 2 such as a lymphokine, which is one part of the polypeptide (I) of the present invention. To 99%.

In the present description, drawings and claims, when amino acids, peptides, protecting groups, active groups, and the like are abbreviated as abbreviations, they may be represented by IU PAC-IUB (Cofflmiseion on Biological Noaenclaure) or in the art. Based on common abbreviations in

OMPI It is listed in the table. When there is an optical isomer for amino acids, etc.)), the L-isomer shall be indicated unless otherwise specified.

Table 1

D N A Deoxyribonucleic acid

A adenine

T Timmin

G Guayyun

C cytosine

R N A Ribonuclear 黢

dATP Deoxyxyadenosine triphosphate

dTTP deoxythymidine triphosphate

dGTP Deoxyguanosine triphosphate

dCTP Deoxycytidine triphosphate

ATP adenosine peracid

EDT A Ethylenediaminetetraacid

S D S Dodecadium

G 1 y Glycine

A l alanin

Y 1 Norrin

L e u Roy Shin

l i e iso σ

Ser Serine

T h r

C y β cystine

M e G 1 u: glutamate

A s: Asbalaginic acid

Lys: Resin

A r g: Agin

H i s: Histidine

P h β: Fenriya = n

T r: H

Τ r ρ: Tributane

Pro: brolin

A s η: Asparagine

O l: Gutamin

<Q1 η: Pylog terminal

In the present box, the method of releasing the Z-dew as the antiviral activity (IFii-r activity tt) of the polypeptide was carried out in the following section. I-a and crude IFN- _r derived from leukocytes were determined using a test to inhibit the cytopathic effect of VSV against FL cell lines derived from human amniotic membrane. determine the value was standard for I ί ¹ N one r. Meniwa was mosquito value calculation of Poripe Buchido in资料of interest, always systems of this standard IFN-gamma Sorting by the aforementioned WISH- VSV Atsushi was carried out, and power was calculated from the ratio.

Note that the transformant J. coli (disclosed in the Examples below)

Escherichia coli) 294 / pHIT tr 1101 -d 2 was deposited at the Institute for Fermentation, Osaka under the accession number IF 0-143 550 and traded on June 6, 1984. Accession number: PERUP -7658 to the Microbial Industry Research Institute (FR) of the Ministry of Industry

ΟΜΡΙ Has been deposited respectively.

Also, the transformant Escherichia coli 294 / pHITtrl 201-d4 was deposited with the Fermentation Research Institute under the deposit number IFO-14365 from September 4, 1980. O Deposited with the Microorganisms Research Institute of the Ministry of Industrial Science and Technology under the accession number FERM P-7828.o

Brief description of drawings

Figure 1, ^ 2 ^ 1. Brother 3, Figure 4, and Figure 5 show Example 1 (、),

(ϋ). The plasmids shown in (iii) and (iv :) pHITtrp 1101 -d 2 · HI trl 201-d 3, pHI tr 1201— d 4 · pHIT trp 1201 and pHIT trp l 201— d 4 FIG.

BEST MODE FOR CARRYING OUT THE INVENTION

1) According to the example below! The present invention will be described more specifically, but the present invention is not limited thereto.

Example of production 1 Transformant production

(|) IFN-r expression plasmid PS IT trpl 101 [EPC Publication No. 0103898 Reference Example 2 (Jiangsu) Ginseng] digested with restriction enzymes AvaH and Pstl —: Pst 1 kb DNA fragment was isolated. Oligonucleotide adapters containing the protein-synthesis BI initiation codon chemically synthesized by the aforementioned ester method are added to this DNA fragment.

CGA AATGTGCCAG

TATTACACGGT CCTG

Was ligated to the]] portion of AvalE using T 4 D If A ligase. Plasmid ptrp 771 (see Reference Example 2 (I) in the above publication) was cleaved with restriction enzymes Clal and Pstl, and the IPN-r gene obtained by cutting the adapter was connected to the downstream of the trp promoter of the DNA fragment obtained.揷 Enter, DN ▲ [! ? ; However is TGCCAG, Z ¹ has a ¾ group sequence (V)], volume Li Bae Buchido [gamma; expression where the Cys ~ Gln, Z encodes Bae Buchido (Summer)] Burasumido pHIT trpl 101 - d2 (Fig. 1).

Escherichia coli 294 was transformed with the plasmid pHIT trpl 101-d2 according to the method of Cohen et al. The transformed Escherichia coli (E. coli) 294 / pHITtr101-d2 was obtained.

. (Vertical) limits the Example 1 (1) in the same manner as IFN one _r expression Burasumido PHITtrp 1101錚素AvaH was digested with Pstl, IFN - Avail containing Γ gene portion - Pst to preparative 1 k-to DNA fragments min. Oligonucleotide adapter containing a protein synthesis initiation codon synthesized by the above-described triester method with this DNA fragment.

AA TTC ATG C AG

GT ACGTCCTG

Is bound to the back of Avail using T4D Mf A ligase. Separately, the current vector ptr _P 7oi [Reference Example 2 of the above-mentioned publication] (im was digested with the restriction enzyme EcoRI, then partially degraded with Clal, and the resulting margin was repaired with DNA volimerase I large fragment. 4 The expression vector ptrp781 was constructed using a DNA ligase to form a circular form, the ClaI recognition site closer to the EcoR I recognition site was broken, and the insertion site for the heterologous gene was E (3 ORI sites).

The adapter-linked IFN-r gene is inserted downstream of the tributophan promoter of a DNA fragment obtained by digesting Ptrp781 with the restriction enzymes EcoRI and Ps11, and is ligated using T4DNA ligase. I, DNA CIV; however I ¹ is CAG, Z ¹ has a base S column (V)], Boripe Buchido [1 '; however is Gln, Z is expressed Burasumido pHITtrpl encoding Bae Buchido ([pi)] 201—d3 can be constructed (Figure 2). The plasmid pH ITtrpl 201-d3 is used to transform E. coli 294 according to the method of Cohen et al. (Supra) to obtain a strain E. coli294 / pHITtrp 1201 to d3 containing the plasmid. Example 1 In the same manner as in Example 1 (I), IFN-r expression plasmid PHIT trpl101 was digested with restriction enzymes Avail and PStl, and Aval-Pstl 1 kb containing the IFN-fit gene portion was digested. Separate the D If A fragment. The D margin generated by the digestion of this DNA fragment with the restriction enzyme Ava! E]]) After filling in using NA polymerase I large fragment, the protein synthesis starting point was chemically synthesized by the Trieste method. Oligonucleotide linkers containing codons

C ATGAATTC ATG

With T 4 D! Ί A ligase.

! This turned into anti restriction enzyme EcoRI IFN-r gene by binding linker, cleaved with the restriction enzymes Ec EcoRI and Pstl by the be inserted downstream of Toributofu Umbro乇one coater of ptrp781, DNAC IV; however Y ¹ Is a bond, Z ¹ has a base sequence (V)], and polypeptide !: Γ; Y 'is a bond, and Z is an expression plasmid pHI T trpl 201-d encoding peptide (I)]. Can be constructed (Fig. 3).

The plasmid pHIT trpl 201-d4 is used to transform Escherichia coli 294 according to the method of Cohen et al. (Supra), and the E. coli 294 / pH IT trp 1201 to d47 containing the plasmid is obtained. .

one.

wi? oj -iY- (iv) Plasmid containing IFN-r gene pHIT3709 (see fiPC Publication No. 0103898, Reference Example l (Vii)) was partially digested with the restriction enzyme BstNI to obtain BstNI-Pstl fragment. I got it. Oligonucleotide adapter containing the chemically synthesized protein synthesis initiation codon ATG at this BstNI cleavage site

AATTCATGTGTTATTGTC GTACACAATAACAGT

Was ligated using T4 DNA ligase.

On the other hand, the plasmid pt rp78 treated with EcoRI and PstI was ligated. The IFN-r gene was ligated using T4 DNA ligase to construct an IFN-r expression plasmid pHIT trpi 201 (FIG. 4).

Digest pHIT trp 1201 with restriction enzymes Ava H and Pst I and contain 311-? A 311 l kb DNA fragment was collected.

After repairing the AvaΠ-cleaved portion of this DNA with DNA polymerase gel fragment, the oligonucleotide adapter 1 (CATCGATG) containing the protein synthesis initiation codon synthesized by the triester method was used using T4 DNA ligase. To the restoration. The IFN-r gene thus obtained was inserted downstream of the ptrp 77 1 trp promoter cut with Cla I and Pst I obtained in Example 1 (ί :), and the polypeptide [I; However, the expression plasmid pHITt 卬 1201-d4 encoding Y is a bond and Z is a peptide (M)] was constructed (Fig. 5).

Escherichia coli 294 was transformed with the plasmid pHIT t 卬 1201—d4 according to the method of Cohen et al. (Supra) to obtain a strain containing the plasmid E. coli 294 ZPHIT trp 120 ld4. Was.

ΟΜΡΙ

WIPO »- Example of culture 2 Transformant culture

(I) Escherichia coli 1 A strain containing E. C011294 / pHI Ttrp 110 l-d2 constructed in (I) containing 8 μ9, ηί tetracycline, 0.4% casamino acid, 1% glucose The cells were cultured in 37 medium using 9 medium, and when the growth reached KU220, 3 indolylacryl I (IAA) was added to 25 μ9 / ml, and the cells were cultured for another 4 hours. After the culture, the cells were collected by centrifugation and suspended in 0.05 M Trie-HC11 1> 37.6 containing 1Z10 of 10% sucrose. To this suspension was added 1 mil, α 2, 10 mM, phenylmethyl sulfide, NaCl, ethylenediamine tracetate (EDTA), spemidine, and lysozyme, respectively.

In addition to taking 40 mlt bottles 200 / *?) ^, The cells were placed at 0 for 1 hour and then treated with 37 for 3 minutes to obtain a lysate.

-This lysate was centrifuged for 30 minutes in a 4 centimeter, 20000 rp heavy (server centrifuge SS-34 rotor), and the polypeptide [I ·; X was a bond or (and) Met, Y Obtained a supernatant containing Cys-Gin and Z peptides (summer)]. The antiviral activity of this supernatant was 2.87 × 10 ⁸ U / ^ culture solution.

(I) Example 1 (Surface) Tfi transformant E. coli 294 / pHI trpl 201-d3 was cultured and extracted in the same manner as in Example 2 (I), and the polypeptide [I; A bond or (and) Met, Ϊ is Gin or (and) <Gln, Z is a peptide (X)]-containing compound.

The anti-viral activity of the above was determined, and a plant equivalent to that of Example 2 (I) was obtained.

(Hidden) The transformant obtained in Example 1 (II) was cultured and extracted from E. coli 294 / pHI trpi 201—d4 'in the same manner as in Example 2 (I).

OMPI — 丄 g—

(I, where X is a bond or (and) Met, Y is a bond, Z is a peptide (H))-containing supernatant.

Measure the antiviral activity of this supernatant and obtain the same value as in Example ² (i) .o

(IV) The transformant E. coli 29 pHITtrp 1201 d4 obtained in Example 1 (iv) was cultured and extracted in the same manner as in Example 2 (i), and the polypeptide [I; Hand or (and) Met, Y is a bond, Z is a peptide

(1)] content was obtained. When measuring the anti-viruses activity of this supernatant was ^2. ^⁵ X 10 ⁵ UZ culture.

Example 3 Purification of polypeptide obtained by guanidine hydrochloride extraction

(I) The bacterium 5.9- obtained in the same manner as in Example 2 (I) contains 7 M-inoculated guanidine and 2 mil phen-methyl sulfo-fluoride (XI M tris Suspend in loose mouth liquid (pH 7.0) 1, stir at 4Τ3 for 1 hour * Centrifuge at 10,000 X for 30 minutes to obtain supernatant 2 to obtain io supernatant, 137 sodium sodium oxalate, 2 · 7 aM Potassium potassium, ai all Lactate (pH 7.4) consisting of lina sodium phosphate and 1.5 all monobasic phosphate (referred to as PBS below) 26 Mor2—11.1, applied at a flow rate of 1 · / min to the column volume 1. Then, the column was filled with (X5 @ 20 g sodium phosphate buffer solution (PH 7.0) containing guanidine hydrochloride (PH 7.0) 60 »/). After washing, 3 eeit of 20-fold M sodium phosphate bite solution (pH 7.0) containing 2M guanidine diacid was taken out to obtain a fraction having anti-viral activity.

Preliminarily, this area 2 was previously treated with a 25 mM ammonium phosphate buffer solution (pH 6.0) containing 1 mM ethylenediaminetetraacetic acid, 15 mM sodium chloride, 10 aM cysteine and 2 M guanidine. It is applied to a column of Sepafari S-200 (Famar) made into a flat mouth (2.6 x 9 <», column capacity 500 W), eluted with the same mouth liquid and has anti-discharge activity. Min 37 W.

The polypeptide obtained here [I; X X is a bond or (and) Met, Ϊ is Cya-Gin, Z is vetide (S)] is 5.9 W]. Specific activity is (L 0 was X 10 ^7. in this preparation the Laemali method [Nature,

227, 680-685, (1970) 3, analysis of sodium dodecamate polyacrylamide gel by electrophoresis showed that the mature form of I. If Nr [US Patent Application No. 53404 (filing date, September 1983) 20 —Zi—

(See the description on the day) and the specification), a protein band was detected at a position showing almost the same mobility (molecule 惫 about 18,000). On electrophoresis under non-reducing conditions, a slight protein band was observed at a position corresponding to the molecular weight of the dimer. That is, the formation of Beni-mer was much less than that of the traditional rl FN-Nicara.

(I) Example 2 The frozen S-isomer obtained by the method of (I) and (■) was added to 0.1 M each containing 7 M guanidine and 2 mM phenyl methoxide;! Suspend in 3 times the volume of Tris-HCl buffer (pH 7.0), stir at 4 for 1 hour, and centrifuge at 100,000 X for 30 minutes. The upper part is diluted 14 times with PBS and applied to the antibody column (ΜΟ Γ2-11. 1). The column is then washed with (20 BU sodium phosphate gent containing a solution of guanidine dioxate (X 7.0)) and then washed with 20 mM sodium phosphate containing guanidine diacid. The mixture is shaken with a buffer solution (pH 7.0) to obtain a compound having antiviral activity, and the fraction is prepared in advance using 1 m diethylene diamine tetraacetic acid. 0.15 M sodium chloride, 10 M cysteine The power of Sephatariya S-200 (manufactured by Fal Macia) equilibrated with 25-BM succinic acid-anhydrous acid solution (H 6.0) containing 2M acid guanidine

And elute with the same mouth liquid to obtain a tomato having antiviral activity. Here ^! Each of the resulting polypeptides [I; X is a bond or (and) Met, Y is Gin or (and) Gin, Z is a peptide (summer)] and a polypeptide [I, where X is a bond or (And) Met, Y is an antagonist, Z is a peptide (summer)], and the specific activity is the same as or higher than that of the polypeptide (I) obtained in Example 3 (I).

Example 4 Purification of polypeptide obtained by ultrasonic extraction

Example 25: 25 frozen cells obtained by the methods of (·), (I) and (Jiang) were each 0.15 0.1 sodium sodium borate (pH 9.5) 1.5 times

OMPI Suspend the volume and briefly stir for 1 hour with a 4 wedge, then extract by applying ultrasonic waves 5 times for 30 seconds, and centrifuge at 30,000 × 9 for 1 hour to obtain a supernatant. The supernatant is mixed with 25 W of the previously washed PBS and slowly stirred at 4 t for 1 hour. Thereafter, the silica gel is packed into a column, washed with 20 to 30 times the column volume of 1 M NaCl, and then with 0.01 M sodium borate containing 0.5 M tetramethylammonium chloride. Elution with mild mouth liquid (pH & 0) yields about 20 fractions showing anti-wisdom activity. This was further divided into four fractions, which were applied to a monoclonal antibody (Mor 2-11 1) abundance column equilibrated with PBS, washed with 10 volumes of PBS, and then washed with 50% ethylene. Elute with 20 U sodium phosphate buffer (pH 7.0) containing glycosyl and 1 M sodium chloride. The anti-digestion activity elutes in the first about 20 W. By subjecting the resulting solution containing poly (boloid) (I) to SDS-polyacrylamide gel electrophoresis, the molecular weight of each sample was about 1,500,000 (15 d) and about 17, 0 0 0 (17 K d) band: In each case, the former is the main band.

Those corresponding to the above 15 Kd are each a peptide [I; X is a bond or (and) Met, Y is Cys ^ Gln, Z is Lys], a polypeptide [I; Hand or (and) Met, Y is Gin or (and) Gln, Z is Lys] and polypeptide !: I; X is a bond or (and) Met Y is a bond, Z is Lys] , 17 Kd correspond to the polypeptides [I; where X is a bond or (and) Met, Y is Cy3-Gln, Z is dibutide (H)], polybutide [ I, where X is a bond or (and) Met, Y is Gin or (and) <Gln, Z is peptide (2)]-and polypeptide [I;

OMPI

レ.> WIPO -26- is a bond or (and) Met, Y is a bond, and Z is a peptide (H)].

OMPI WIPO ^ Example 5 Production of polypeptide [I; X is a bond or (and) Met, Y is a bond, Z is peptide (I)]-

(i) The expression plasmid PHIT trp 1201-i4 constructed in Example 1 ((ν) was used to transform E. coli C600 according to the method of Cotien et al. (Escherichia coli = E. coli) C600 / pHIT trp1201-d4 was obtained.

(jj) E. coli C 600 / pHIT trpl 201—d4 was added to 500 ^ © Luria medium (Paktotrypton 10.0 ^, yeast extract 5.0 ^, NaC 5.0 distilled water 1ί). The seeds were inoculated into 37-soil 1 and seed-cultured for 12 hours.

The culture, 9.5 Trp -. 8 Mod 3 medium (sterilized _{_{_{(NH 4) 2 HP0 4 5.0}}} ^ / ^, K 2 HP0 4 6.0 ^ Z, KH 2 P0 4 4.0 i,

_{_{_{NaH 2 P0 4 · Η 2 0}}} 3.0 ^ /, (NH 4) 2 S0 4 2.0 J? / ^ And anti-foaming agent (LB 625), glucose 30 was sterilized separately each ^,

MgS0 ₄ - 7H ₂ 0 0.5, hydrochloride 5 / & thia Mi emissions, Kuen Sanna Application Benefits © beam 0.1, Application Benefits off. To Juan 5 O ^ Z, Tetracycline 5

The mixture was transferred to a 14-ml glass tank containing Cimapec containing).培養 The cells were cultured at 6.6 to 7.0 by adding NH ₄ OH. After 13 hours, IAA was added.

Sampling was performed over time to measure bacterial growth and antiviral activity. The former was measured by absorbance, and the latter was centrifuged and the supernatant was used for the above-mentioned measurement method.

The growth of the bacterium was maximized 14 hours after the start of the culture, and the antiviral activity at that time was 5 × 10 ⁶ U.

ΙΟΜΜ

"WIPO" Industrial applicability

The polypeptide (I) of the present invention has anti-viral, anti-employment and immunological effects, and is stable, so that it can be advantageously used as a pharmaceutical or the like.

O PI

Claims

-26- Scope of request

L expression

(N) HXY-Asp Pro yr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Qly His Ser Asp Val Ala Asp Asn Gl Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Ljs lie Met Gin Ser Gin lie Val Ser Phe Tyr Phe Lys Leu Phe Lya Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Yal Glu Thr lie Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Lys Arg Asp Asp Phe Glu Lye Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala lie His Glu Leu lie Gin Val Met Ala Qlu Leu Ser Pro Ala Al Lya Thr Gly Lys Arg Lys JLrg Ser Gin et Leu Phe Arg Gly-Z— ΘΗ (C)

(Where X is Met or a bond, Y is Cys-Gln, Gln, <Gln or a bond, Z is (N) Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Qly Arg Arg Ala Ser Gin ( C) represents a peptide or amino acid residue having 1 to 16 amino acids counted from its N-terminus in the peptide chain]]. 2. ATG at the 5 'end and downstream

(N) Η-Υ '-Αβρ Pro Tyr Val Lya Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Tal Ala Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Uet Gin Ser Gin lie Val Ser Phe Tyr Phe Lya Leu Phe Lys

O PI —ST—

Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Yal Glu Thr lie Lys Glu λβρ Met Asn Yal Lya Phe Phe Asn Ser Asn Lya Lys Lys Arg Asp Asp Phe Glu Lye Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn 7al Gin Arg Lya Ala lie His Glu Leu lie Gin Val Met Ala Qlu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Qln Met Leu Phe Arg Gly-Z-OH (C)

Wherein Y ′ represents Cys to Gln, Gln or a bond, and Z represents (! I) Lys Arg Lye Arg Ser Gin Met Leu Phe Arg Qlj Arg Arg Ala Ser Qln (C) Represents a peptide or amino acid residue having 1 to 16 amino acids counted from the N-terminus) and a region encoding the polyribobutide represented by A method for producing said polypeptide, comprising culturing the transformant, producing and accumulating the vorivebutide according to claim 1 in the culture, and collecting the vorivebutide.

a At the 5 'end, ATG

(N) HY '-Asp Pro Tyr Yal Lya Qlu Ala Qlu Asn Leu Lye Lys Tyr Phe Asn Ala Gly His Ser Asp Yal Ala Asp Asn Gly Thr Leu Phe Leu Qly lie Leu Lya Asn Trp Lys Qlu Qlu Ser Asp Arg Lys lie Met Gin Ser Qln lie Yal Ser Phe Tyr Phe Lys Leu Phe Lys Aan Phe Lys Aep Asp Qln Ser lie Gin Lys Ser V 1 Glu Thr lie Lys Glu Asp Met Asn Val Lye Phe Phe Aen Ser Asn Lys Lys Lye Arg Asp Aap Phe Glu Lys Leu Thr Aen Tyr Ser 7al Tiir Asp Leu Asn Yal Qln

OMPI Arg Lys Ala lie His Glii Leu lie Gin Val Met Ala Qlu Leu Ser Pro Ala Ala Lys T r Gly Lya Arg Lye Arg Ser Gin Uet Leu Phe Arg Qly-Z-OH (C)

[Wherein represents Cys-01n, Gln or a bond, and Z represents (N) Lye Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gin (C). Represents a peptide or amino acid residue having 1 to 16 amino acids by counting), and a DNA having a translation termination codon.

,, ku, ν, ϊ? Ο

'***** · One ,, -One Summary Form

A novel polypeptide having a biological activity equal to or higher than IFN-r and hardly causing dimerization or multimerization, a transformant containing DNA encoding the novel polypeptide, and a transformant containing the same. A new way to produce new polypeptides from cultures.

The DNA encoding the novel polypeptide can be produced, for example, from a known IFN-r gene (cDNA) -containing plasmid, pHITtrp101 or pHITti "p1201. .

After binding this DNA to a vector, it is transferred to a host to obtain a transformant. An antibody column is used for purification of the polypeptide from the culture.

The new polypeptides produced can be used as antiviral and antitumor agents.

ΟΜΡΙ

W1PO *