DK175337B1 - Method for Preparation of a Polypeptide with Hirudin Activity and DNA Sequences, Fusion Peptide, Hybrid Plasmid and Host Organism Useful in the Method - Google Patents

Abstract

Hirudin can be obtained using a specific DNA sequence in a genetic engineering process. The gene is advantageously synthesised in the form of several fragments which are enzymatically ligated to give two larger part-sequences which are incorporated into hybrid plasmids and amplified in host organisms. The part-sequences are reisolated and then combined to form the complete gene which is incorporated into a hybrid plasmid and the latter is expressed in a host organism. <IMAGE>

Description

DK 175337 B1 Λ

The present invention relates to a method for producing a polypeptide with hirudin activity and DNA sequences, fusion peptide, hybrid piasmid and host organism which can be used in the method.

Hirudin is a polypeptide recovered by Hirujid midicinalis and which exhibits a specific antithrombiric activity and acts as an anticoagulant.

Natural hirudin at position 63 contains a sulfated amino acid, Tyr-O-sulfate, cf. J. Dodt et al., FEBS 10 Letters Vol. 165 (2), 180-184 (1984). It is known that the activity of hirudin decreases with increasing desulfation, cf.

JY Chang, FEBS Letters Vol. 164 (2), 307-313 (1983). Thus, for 100% desulfated hirudin, the activity is less than half that of natural hirudin. Since natural products are the result of a long evolution, it should not be assumed that the sulphate group can be renounced. Thus, for one of ordinary skill in the art, there has been no need to manufacture the desulfate product genetically and examine this for hirudin activity. Admittedly, one of ordinary skill in the art would have regarded the desulfate protein as an intermediate for the natural product. However, since no methods are known for the targeted sulfation of such an intermediate by the tyrosine residue, one skilled in the art would again have waived such considerations.

However, surprisingly, it has been found that the polypeptides of the general formula I listed below, which are prepared genetically and are therefore not sulfated, have a biological activity of about the same size as that of natural hirudin.

The invention thus relates to a method for the future

position of a polypeptide having hirudin activity of general formula I

Η- (X) m_ in -X1-C-Tyr-D-Asp-Cys-E-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly -Ser-Asn-Val - Cys -Gly-Gln-Gly-Asn-Lys-Cys-35 Ile -Leu-Gly - Ser-Asp - FG-Lys - Asn-Gln-Cys-Val -Thr-Gly-Glu-Gly-Thr-Pro-Lys -Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-H- I DK 175337 B1

B Ile-Pro-Glu-Glu-Tyr-Leu-Gln- (Z) n-OH I

where m is 0-50, I

B n is 0-100 and I

B 5 X and X1 are the same or different residues of genetic 'I

B encodable amino acids, I

B C is Thr, Val, Ile, Leu or Phe, I

B D is Thr, I

B E is Thr, I

B 10 F is Gly, I

B G is Glu, I

B H is Glu and I

B Z is the same or different residue of genetic I

B encodable amino acids, I

B 15 with the exception of polypeptides in which I

B a) X1 and C are both Val, I

B b) X 1 is Ile and C is Thr, or

B c) where N-terminally amino acids are eliminated until position I

In i0,

B 20 which method is peculiar in that in an ex

B expression plasmid incorporates a gene encoding a polypeptide

B time of the formula I or partial sequences thereof or for an I

B fusion peptide consisting of a polypeptide of formula I or I

B partial sequences thereof and the amino acid sequence I of the β-galactosidase

B 25 in whole or in part, I

and the gene is expressed to form a polypeptide of Form I

I or a fusion protein consisting of a polypeptide with I

In the formula I and the amino acid sequence of the β-galactosidase completely or I

partially and a resultant fusion protein is cleaved to form I

I of a polypeptide of formula I.

In addition, the invention relates to: I - DNA sequences I, IA, IB, IIA and IB listed below, - a fusion peptide which is characterized in that it consists of a polypeptide as defined above and β- galacto- In the amino acid sequence of the sidase, in whole or in part, a hybrid plasmid which is characterized in that it contains a DNA sequence encoding a polypeptide as defined above and a host organism, preferably E. coli, which are properties -5 is similar in that it contains a hybrid plasmid as defined above.

The necessary gene can be chemically synthesized by known methods, isolated from the genome and processed, or the mRNA isolated from induced cells by known methods and the cDNA recovered therefrom.

The pathway over cDNA is the preferred and above all chemical synthesis, especially according to the phosphite net.

Furthermore, synthesis of a gene encoding the polypeptide of formula I, wherein m is 1 and n is 0, X A is preferred.

15 is Met or Arg and C is Thr. A further preferred embodiment of the invention relates to polypeptides of formula I wherein H- (X) m - ^ X- -C- is not H-Val-Val- when n is 0.

Particularly preferred is the synthesis of polypeptides of formula I wherein m is 1, X1 is Met or Arg and n is 0. The preferred DNA sequence for this is as follows:

I DNA Sequer and I I

I Triplet No. '0 1 2 3 4 5 I

In Amino Acid With Tyr Tyr Thr Asp Cys I

Nucleotide No. 1 10 20 I

In Code. strand 5 'CT AGA ATG ACG TAT ACT GAC TGC I

Non-meat. string 3 '. T TAC TGC ATA TGA CTG ACG I

I 6 7 8 9 · 10 11 12 13 14 15 I

Thr Glu Ser Gl'y Gin Asn Leu Cys Leu Cys I

30 40 50 'I

ACT GAA TCT GGT C AG AAC CTG TGC CTG TGC I

TGA CTT AGA CCA GTC TIG GAC ACG GAC ACG, I

I 16 17 18 19 20 21 22 23 24 25 I

H Glu Gly Ser Asn Val Cys Gly Gin Gly Asn I

60 70 80 I

GAA GGA TCT AAC GTT TGC GGC CAG GGT AAC I

CTT CCT AGA TIG C AA ACG CCG GTC CCA TIG I

I 26 27 28 29 30 31 32 33 34 35 I

I Lys Cys Ile Leu Gly Ser Asp Gly Glu Lys I

90 100 110 I

AAA TGC ATC CTT GGA TCC GAC GGT GAA AAC · I

ITT ACG TAG GAA CCT AGG CTG CCA CTT TIC I

I 36 37 38 39 40 4l 42 43 44 45 I

M Asn Gin Cys Val Thr Gly Glu Gly Thr Pro I

120 130 140 I

AAC CAG TGC GTT ACT GGC GAA GGT ACC CCG I

TIG GTC ACG C AA TGA CCG CTT CCA TGG GGC I

I 46 47 48 49 50 51 52 53 5 4 55 I

I Lys Pro Gin Ser His Asn Asp Gly Aso Phe I

150 160 170 I

AAA CCG CAG TCT CAT AAC GAC GGC GAC TIG I

TIT GGC GTC AGA GTA TIG CTG CCG CTG AAC I

I 56 57 58 59 60 61 62 63 64 I

I Glu Glu Ile Pro Glu Glu Tyr Leu Gin Stp I

l80 190 200 I

GAA GAG ATC CCT GAG GAA TAC CTT CAG TAA I

I CTT CIC TAG GGA CTC CTT ATG GAA GTC ATT I

I Stp I

I 210 I

TAG AGC TCG 3 'I

ATC TCG AGC AGC T 5 '“I

DK 175337 B1

ISLAND

5

As you know, the genetic code is "degenerate", ie. there is only a single nucleotide sequence encoding two amino acids, while the other 18 genetically encoded amino acids can be encoded with -2-6 triplets. In addition, the v; * rts-5 cells of different species do not always make the same use of the variants provided. Thus, there is an unmistakable diversity of codon possibilities for the synthesis of the genes.

It has now been found that DNA Sequence I, which corresponds to the total amino acid sequence, as well as the DNA part sequences IIA used in the synthesis of Sequence I, eg 113 DNA Seouens II A (HIR-I) 15a

Nucleotide no. 10 20

Coding rod 5 * CT AGA ATG ACG TAT ACT GAC

No-meat. string 3 'T TA C TG C ATA TG A CTG A

Xbal 20 I b I c 30 40 50

ACT GAA TCT GGT CAG AAC CTG TGC CTG TGC

TGA CTT AGA CCA GTC TT G GAC ACG C-AC ACG

25

Ib Id I c le 60 70 80

GAA GGA TCT AAC GTT TGC GGC CAG GGT AAC

30 CTT CCT AGA TTG CAA ACG CCG GTC CCA TTG

Id If I e 90 35 AAA TGC ATC CTT G Child HI 3 'TTT ACG TAG GAA CCT AG 5' I f I DK 175337 B1 i ° DNA Sequence TI B (HIR-II) I II a 5 Nucleotide No. 100 Hq

In Coding String 5 'GA TCC GAC GGT GAA AAG

In Non-code. string 3T BamHI G CTG CCA CTT TTC

I II b I

I II and II c I 10 120 130 mo

I AAC C AG TG C GTT ACT GGC GAA GGT ACC CCG

TTG GTC ACG CAA TG A CCG CTT CCA TGG GC-C

I II t II d I

II c le I 15 150 160 iγο

I AAA CCG C A G TCT CAT AAC GAC GGC GAC TTC

I TTT GGC GTC AGA GTA TTG CTG CCG CTG AAG

I II d II f II e II g I 20 180 190 200

I GAA GAG ATC CCT GAG GAA TAC CTT CAG TAA

CTT CTC TAG GGA CTC CTT ATG GAA GTC ATT

I II f Uh I II g I 25 210 TAG AGC TCG Sal 13 'ATC TCG AGC AGC T 5' I 11 h I 30 a particularly advantageous for genetic engineering synthesis is the particularly preferred form of hirudin. At the 5 'end I of that incocency strand in DNA sequence I, an unwanted DNA sequence corresponding to restriction endonuclease is read an XbaI; At the 3 'end of the stranding strand therein is found against the single-stranded protruding sequence corresponding to the restriction enzyme Sal I.

ISLAND

7 DK 175337 B1

recognition sequences ensure insertion of DNA into plasmids of the desired orientation. (However, it is also possible to select the same recognition sequences and make a similar selection after characterizing S

the plasmid DNA by corresponding restriction or DNA sequence analysis or by expression).

Between these recognition sequences and the codons for the amino acid sequence are found in the 5 'end coding codon for the amino acid methionine (as in the DNA sequence I. is designated 0). Alternatively, a pre-sequence (also called signal or leader sequence) of a bacterial or other host protein can be found (review article: Perlman and Haivorson, J. Mol. Biol.

167, 391 (1933) 3, which causes secretion of the desired pclypeptic from the cytoplasm, cc which is cleaved by this secretion process from a signal peptidase naturally occurring in the host cell. At the end of the inc coding strand, according to the invention, after stopping for glutamine coke, a stop cocoon or preferably - as prepared in DNA sequence I - two stop triplets. In addition, between these stop codons and the protruding Sall enzyme, the DNA sequence is incorporated into a nucleotide sequence corresponding to the restriction enzyme SstI.

An internal singular section of the restriction enzyme Bam HI (in codon 20/31) allows subcloning of two gene fragments HIR-I and HIR-II (cf. DNA sequence II), which can be incorporated into well-studied cloning vectors, such as pBR 322 , pUC 8, or pUC 12. In addition, a number of additional singular recognition sequences are incorporated into the gene by enzymes which, on the one hand, provide access to sub-sequences of hirudin and, on the other hand, allow variations to be made (Table I): 35 I DK 175337 B1 I 8

In Table I I

Restrikt. Cut after. nucl. No. Restricted. Sn. eft. nucl.

In enzyme_ (encode, string) _enzvm_nr. (encode, size)

Rsa Ia) 8- · Hph I 117

I 5 Acc I 12 Rsa I ^ 137 I

Hinf I 27 Κρη I 139.

I Xho IIa) 57 Taq I 172 I Fnu 4 HI 71 Xho IIb) 178 I Hae III 73 Dde I 184 I 10 Bst NI 75 Mbo II 136

Sst I 210

I a) singular with respect to part sequence HIR-I I

In b) singular with respect to sub-sequence HIR-II I

In the DNA sequence I can be made up of 14 oligonucleotides

In times with a length of 25-35 nuclectides (cf. DNA Se- I

In sequence II), first chemically synthesized

I and then via "sticky ends" of 4-6 nucleotides

20 is enzymatic. IN

In the DNA sequence I, it should also be noted,

In that for the amino acids that can be encoded with multiple co- I

In donors, these are not equivalent, but on the contrary, I in the host cell concerned, such as E. coli, differ

In 2 ^ preferences. In addition, palindromic sequences I are reduced

to a minimum.

Therefore, the DNA sequence I gene structure is readily available from relatively small building blocks, allowing subcloning of two gene fragments into well-known vectors and allowing them to be combined into a single gene and making any changes to it.

Depending on the incorporation of the synthetic I gene into the cloning vector, the desired peptide I is expressed directly with hirudin's amino acid sequence (DNA sequence I 35 IC) or in the form of a fusion protein with a bacterial I protein such as (3-gaiacosicase or as partial sequences DK 175337 B1

ISLAND

9 of these. Such fusion proteins can then be chemically or enzymatically cleaved in known manner. Is there for example. at position 0 in the sequence I of the amino acid methionine (DNA sequence 1a), then a chemical cleavage can occur with bromocyanine; but if in this position the amino acid is arginine (DNA sequence IB), then an enzymatic cleavage can occur with trypsin.

DNA sequence IA: 10 0

Met (Amino Acids 1-64) 1 5 205 210 5 'CT AGA ATG (Nucleotide. 9-200) TAA TAG AG C T 3' t5 3 'T TAC (complement, nucl.) ATT TAC 5'

Xba

Sst

DNA Sequence IB: 20

ISLAND

Arg (Amino Acids 1-64) 1 5 205 210 5 'CT AGA CGT (Nucleotide. 9-200) TAA TAG AGC T 3' 25 3 'T TAC (complement, nucl.) ATT ATC 5'

Xbal SstI

DNA Sequence IC: 30 0

Met (Amino Acids 1-64) 205 210 5 'AA TTC ATG (Nucleotide' 9-200) TAA TAG AGC TCG 3 'G TAC (complement. Nucl.) ATT ATC TCG AGC AGC T 5' 35 EcoRJ Sal

I DK 175337 B1 I

I '10 I

Variations of the amino acid sequences may occur according to sara I

replacing the synthetic gene at the DNA level by replacement I

Recalling the corresponding gene fragments with newly synthesized I

H DNA sequences utilizing the corresponding restriction I

5 enzyme cross-sectional sites. IN

As an example of a variation of the amino acid sequence, I can

are mentioned the polypeptide of formula I (known), wherein m is 1 and I

X1 and C are Val and n is 0. This modification can easily be done I

via the cut site corresponding to the restriction enzyme Acc I. IN

10 The incorporation of the synthetic gene or gene I

H fragments in cloning vectors, e.g. those in the trade being- I

In the plasmids pUC 8, pUC 12 and pBR 322 or other generic I

In partially accessible plasmids such as ptac 11 and pKK 177.3, I

You happen in a manner known per se. The chemical synthesis- I

15 genes can also be provided with suitable chemical I in advance

synthesized control regions that enable an express I

sion of the proteins. In this connection, I-

See the Maniatis textbook (Molecular Cloning, Maniatis I

In Others, Cold Spring Harbor, 1982). The transformation of I

The hybrid plasmids thus obtained in suitable host organ I

In niches, preferably E. coli, are also per se

In known and described in the above textbook in detail. IN

In the recovery of the expressed protein and its purification

Curing can be done by known methods. IN

I 25 I

The invention will be explained in the following

In detail in examples by means of some further explanations

forms of leadership, many of which are possible changes and I

Combinations will be apparent to those skilled in the art. Percentage I

For 30 watches, here is by weight, unless otherwise stated. IN

Example 1 I

Chemical synthesis of a single-stranded oligonucleotide I

With the rebuilding block Ia comprising nucleotides I

35 ne 1-32 in the encoding string, as an example I will now

the synthesis of the rebuilding stones is explained. Using I

of known methods [M.J. Gait et al., Nucleic Acids Res. IN

11 DK 175337 B1 8, 1080-1096 (1980)) binds the nucleoside present at the 3 'end, in the present case thymidine (

D

cleotide No. 32) to silica gel ("Fractosil", Company Merck) covalently via the 31-hydroxy function, thereby first reacting the silica gel with the decomposition of ethanol with 3- (tri-ethoxysilyl) propylamine to produce Si-O-Si The thymidine is reacted as 31-O-succinoyl-5'-dimethoxy-oxytrityl ether in the presence of paranitrophenol and N, N'-di-cyclohexylcarbodiimide with the modified carrier, whereby the free carboxy group in the succinoyl group acylates the amine propylamino group.

In the following synthesis steps, the base component is used as 5'-O-dimethoxytrityl nucleoside-3'-phosphoric acid monomethyl ester dialkylamide or chloric, the adenine being N-benzoyl compound, cyto-4 * 2 sinet as N -benzoyl compound, the guanine as N-isobutylryl compound, and the thymine containing no amino group are present without a protecting group.

50 mg of polymeric carrier containing 2 µmol of thymidine are treated in the order of: (a) nitromethane, (b) saturated zinc bromide solution in nitromethane with 1% water, (c) methanol, 25 (d) tetrahydrofuran , (e) acetonitrile, (f) 40 µmol of the corresponding nucleoside phosphite and 200 µmol of tetrazole in 0.5 ml of anhydrous acetonitrile (5 minutes), (g) 20% acetanhydride in tetrahydrofuran with 40% lucidine and 10% dimethylaminopyridine (2 minutes), (h) tetrahydrofuran, (i) tetrahydrofuran with 20% water and 40% lutidine, (j) 3% iodine in collidine / water / tetrahydrofuran in volume ratio 5: 4: 1, I DK 175337 B1

(k) tetrahydrofuran and I

(1) methanol. IN

As used herein, "phosphite" means deoxyribose-3'-monophosphoric acid monomethyl ester, the third vein being saturated with chlorine or a tertiary amino group, e.g. a morpholine residue. The yields from the individual synthesis steps can be determined spectrophotometrically by the detritylation reaction b) by measuring the absorption of the dimethoxytitrile cation at a wavelength of 496 nm.

After completion of synthesis of the oligonucleotide 10, the methyl phosphate protecting groups of the oligomer are cleaved by p-thiocresol and triethylamine.

H The oligonucleotide is then cleaved off by treatment with ammonia for 3 hours from the solid support. A treatment of the oligomers for 2-3 days with concentrated 15 ammonia quantitatively cleaves the amino protecting groups of the bases. The crude product thus obtained is purified by H high pressure liquid chromatography (HPLC) or by polyacrylic H amide gel electrophoresis.

H Quite similarly, the other gene-building blocks Ib-IIh are also synthesized, whose nucleotide sequence appears in DNA sequence II. 1

Example 2

Enzymatic binding of the single-stranded oligonucleotides

| times to the gene fragments HIR-I and HIR-II

For phosphorylation of the oligonucleotides in the 5 'end position, each nmol of the oligonucleotides Ib-Ie is treated with 5 nmol adenosine triphosphate with four units of T4 polynucleotide kinase in 20 µl of 50 mmolar Tris-I 50 HC-1 buffer ( pH 7.6), 10 mmolar magnesium chloride and 10 mmolar dithiothreitol (DTT) for 30 minutes at 37 ° C.

In the Enzyme, deactivate at 95 ° C for 5 minutes.

The oligonucleotides Ia, If, Ila and Uh, which in the DNA sequence Η IIA and IIB constitute the protruding sequences, are not phosphorylated in I 35. This, in subsequent ligation I, prevents the formation of larger sub-fragments than those that correspond to DNA sequence IIA or IIB.

13 DK 175337 B1

The oligonucleotides Ia-If are ligated as follows to sub-fragment HIR-1: 1 nmol of each of the oly gonucleotides Ia and If as well as of the 5 'phosphates of Ib /

Ic, Id and le are dissolved together in 45 µl of buffer containing 50 mmolar Tris-HCl (pH 7.6), 20 mmolar magnesium chloride, 20 mmolar potassium chloride and 10 mmolar DTT.

To bind the oligonucleotides of DNA sequence IIA, the oligonucleotide solution is heated for 2 minutes to 95 ° C and then cooled slowly (2-3 hours) to 20 ° C.

10 µl of enzymatic linkage is then added 2 µL of 0.1 molar DTT, 8 µL of 2.6 mmolar adenosine triphosphate (pH 7) and 5 µL of T4 DNA ligase (2000 units) and incubated for 16 hours at 22 ° C.

Analogously, the oligonucleotides Ilb-15 are phosphorylated and then ligated with the oligonouleotides Ila and Uh to the sub-fragment HIR-II.

Purification of. the gene fragments HIR-I and HIR-II are by gel electrophoresis on a 10% polyacrylamide gel (without urea addition, 20 x 40 cm, 1 mm thick), using 20 as the marker substance "ØX 174 DNA" (company BEL), cut with Hinf I or pBR 322 incision with Hae III.

Example 3

Preparation of hybrid plasmids containing the gene fragments HIR-I and HIR-II

(a) Incorporation of the HIR-I gene fragment into pUC 12

The commercially available plasmid pUC 12 is opened in the known manner with the restriction endonucleases Xba I and • Child HI following the directions of the smear. The digested 30 is divided into 5% polyacrylamide gel in known manner by electrophoresis and the bridge piece is made visible by staining with ethidium bromide or by radioactive labeling ("nick translation" according to Maniatis above). The plasmid band 1 is then excised from the acrylamide gel and electrophoretically separated from the polyacrylamide. The digest can be divided into 2% low melting agarose gels (as described in Example 5a).

I DK 175337 B1 I

I 14 I

In 1 µg of plasmid is then ligated with 10 ng of gene fragment I

In HIR-I, as described in Example 2, phosphoryl-I

In dish, overnight at 16 ° C. Thereby the hybrid plasmid, I

Referring to FIG. 1. I

I 5 (b) Incorporation of the HIR-II gene fragment into pOC 8 I

By analogy with (a) it is opened commercially I

In plasmid pUC 8 with Barn HI and Sal I and ligated with gene frag-I

In the ment HIR-II, as described in Example 2, is pnos- I

In phorylated. Hereby it is shown in the drawing FIG. 2

In 10 hybrid plasmid. IN

In Example 4 I

I The synthesis and incorporation of the complete gene into a plasmid I

Transformation and development

In the hybrid plasmids thus obtained, transform I

Res in E. coli, the strain E. coli K 12 by treatment I

Comply with a 70 mmolar calcium chloride solution

In tent, and the suspension of the hybrid plasmid in 10 mmolar I

In 20 Tris-HCl buffer (pH 7.5) 70 mmolar with respect to I

I to calcium chloride, is added thereto. They transform- I

In those strains, as usual, by selection of I

In the antibiotic mediated by the plasmid I

You are stoning or sensitivities, and the hybrid vectors for I

I 25 meres. After the cells are killed, hybrid plasma I is isolated

In the mites, opens with the restriction I originally used

In enzymes, and the gene fragments HIR-I and HIR-II are isolated

I by gel electrophoresis. IN

I (b) Linking of the gene fragments I

I The sub-fragments obtained by propagation HIR-I I

I and HIT-II are linked as described in Example 2 (co-I

In men binding at 60 ° C) enzymatically, and thus obtained I

In synthetic genes with the DNA sequence I are introduced into cloning I

In the vector pUC 12. This yields the hybrid plasmid of Fig. I

In FIG. 3. I

(C) Preparation of a gene for [Val, Val] -hirudin.

From the plasmid of FIG. 3 is thus obtained as described in Example 5 (a) the AccI-SalI fragment (nucleotides 13-212), which then by means of the adapter 5 5 'CT AGA AT G GTT GTA T 3' 3 'T TAC CAA CAT ATA 5 '

Xbal Accl can be ligated into the plasmid .pUC 12, which has already been opened with Xbal and SalI.

10

Example 5

Construction of hybrid plasmids for expression of DNA-15 sequences IA, IB and IC

(a) Incorporation of DNA sequence IC into pKK 177.3 (direct expression)

The expression plasmid pKK 177.3 [plasmid ptac 11, Amman et al., Gene 25, 167 (1983), where a sequence containing a Sal-I cut site is synthetically incorporated into the Eco-RI gene 20 site] with the restriction enzymes Eco RI and Sal I. From the plasmid shown in FIG. 3, the DNA sequence IC is recovered as follows: The plasmid is first cut with the restriction enzyme Sal I, then with the enzyme Acc I and then the small Acc I-Sal I fragment is separated by gel electrophoresis on a 2% The low melting agarose gel from the plasmid band, recovering the DNA by dissolving the gel at elevated temperature (according to the manufacturer's instructions) - This DNA fragment can, by means of the following α-daptor 5 'AATTC ATG ACG T 3' 3 'G TAC TGC ATA 5'

Eco RI Acc I

2 is converted to the DNA sequence IC.

By ligation of the cut plasmid pKK 177.3 with the gene IC, a hybrid plasmid is obtained in which an expression of

In or regulatory region is deposited before the inmate. IN

After the addition of a suitable inductor such as isopropyl-β-I

In -thiogalactopyranoside (IPTG), a mRNA leading to I is formed

I for expression of the methionyl polypeptide corresponding to I

In the DNA sequence IC. 'IN

I (b) Incorporation of the hirudin gene IA or IB into ex- I

In the expression plasmid pCK-5196 (fusion construct) * I

The expression plasmid pCK-5196 (Fig. 1 of the drawing)

I 4) is a derivative of the plasmid pAT 153 (Twigg and Sherrat), I

I 10 which in itself constitutes a "high-copy-number derivative of the I

In known plasmid pBR 322. Det. contains the known Lac I

In the UV ^ promoter with (3-galactosidases) known sequence up to 1

In nucleotide # 1554 (corresponding to amino acid # 518: Trp) I

I R R I

inserted between the tet gene (Hind III from pBR 322) and amp - I

In the 15-gene terminator (Aha III at position 3231 in pBR 322). IN

A further feature of this vector is polyforbine I

In the partial sequence inserted between β-galactosidase I

the sequence and the terminator sequence and which derive from it I

In known plasmid pUC 13. lac-UV ^ promoter transcript I

I R I

20 tion proceeds towards the tet gene. IN

I This plasmid contains at I-galactosidase I

In amino acid # 518 the poly compound Xba Ι-Bam HI-I

I -Sma I-Sst I from the plasmid pUC 13, which acts as clone I

In place of the eucaryotic gene. Plasmid CK-5L96 I

I 25 is opened by the restriction enzymes Xba I and Sst I I

I and ligated with the DNA sequence 1a, which can be isolated from I

I from the plasmid of FIG. 3 using Xba I-I

I -Sst I digestion. A hybrid plasmid is obtained as shown in I

In the drawing FIG. 5, which is behind the lac UV ^ control region

At 30, the codons for the first 518 amino acids hold in β-galac-I

In tosidase and subsequently the codons of Ser-Arg-I

I -Met (hirudin 1-64). IN

In the same way, a hybrid plasmid of type I is obtained

In FIG. 6, which, in plasmid pCK-5196, contains I

In 35 inserts with the DNA sequence IB, however, now codo-I

In the net of the amino acid arginine is placed in front of the codon of I

17 DK 175337 B1 amino acid # 1 (threonine). The DNA sequence IB is derived from the plasmid of FIG. 3, intersecting with the restriction enzymes Sst I and Acc I and ligating with the following adapter: 5 5 'CT AGA CGT ACG T 3' 3 'T GCA TGC ATA 5'

Xba I Acc I

Example 6

Transformation of the hybrid plasmids

Competent E. coli cells are transformed with 0.1-1 µg of the hybrid plasmids containing the sequence I or Ia, 1B or 1C, and deleted for the tac plasmids 1S on ampicillin-containing agar plates for pCK-5196. the hybrid plasmids on agar plates containing tetracycline. Then, clones containing the properly integrated hirudin sequences in the corresponding plasmids can be identified by DNA rapid 2Q reprocessing (Maniatis et al.) - Example 7

Expression of polypeptides showing hirudin activity After transformation of said tac hybrid plasmid into E. coli, a polypeptide is expressed which, in addition to the hirudin amino acid sequence at the amino terminus, also carries another methionyl group, which however can. is eliminated by bromocyanin cleavage. In contrast, after the transformation of E. coli with the hybrid plasmid of FIG. 5 or FIG. 6 fusion proteins of β - galactosidases 518 amino acids and hirudin linked to each other by the sequence Ser-Arg-Met or Ser-Arg-Arg. These fusion proteins can be cleaved with bromocyanine or with trypsin to hirudin and β-galactosidase fragments.

I DK 175337 B1 I

I 18 I

In Example 8 I

Working up and cleaning

I The bacterial strains grown for the desire- I

In the optical density, incubate with a suitable inductor, e.g. IN

In 5 IPTG, for a sufficiently long time, e.g. 2 hours. Then you

The cells are killed with 0.1% cresol and 0.1 mmolar benzyl sulfate

In phonyl fluoride. IN

I After centrifugation or filtration, cell I is absorbed

In the lime in a buffer solution (50 nimolar Tris, 50 mmolar I)

In 10 EDTA, pH 7.5) and mechanically opened, e.g. with a French- I

You? in press or "Dyno-Miihle" ^ - '(company Willy Bachofer, Basel),

In which the insoluble components are centrifuged off. IN

The hirudin-containing product is purified from the supernatant layer

tein using conventional methods. For this, ion-I

In 15 exchange, adsorption, gel filtration columns or depots

In finite chromatography on thrombin or antibody column I

You down. The enrichment and purity of the product are checked by I

By means of sodium dodceyl sulfate acrylamide gel or HPLC-I

In analysis. Fusion proteins with a β-galactosidase fraction I

I 20 can already be detected in the crude extract of the lighted bark

In rows by means of their flow characteristics, which you

You are different from β-galactoseidases (1-518). Characteristics I

In the ring of the technologically produced hirudin you happen

By comparison with that isolated from blood gel, au- I

In 25 tentical substances, possibly using samples based on I

In on hirudin's blood clotting inhibitory properties. IN

I 30 I

I 35 I

Claims (7)

A process for preparing a polypeptide having hirudin activity of the general formula I Η- (X) -C-Tyr-D-Asp-Cys-E-Glu-Ser-Gly-Gln-Asn-Leu-Cys 5 Leu-Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-Gly-Ser-Asp-FG-Lys-Asn-Gln-Cys-Val -Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-H-Ile-Pro-Glu-Glu-Tyr-Leu-Gln - (Z) n-OH 10 where m is 0-50, n is 0-100 and - X and X 1 are the same or different residues of genetically boring amino acids, C is Thr, Val, Ile, Leu or Phe, 15. is Thr, E is Thr, F is Gly, G is Glu, H is Glu, and 20. are the same or different residues of genetically encoded amino acids, with the exception of polypeptides wherein a) X1 and C are both Val, b) X1 is Ile and C is Thr, or c) N-terminally eliminated amino acids up to position 10, characterized in that an expression plasmid incorporates a gene encoding a polypeptide of formula I 'or partial sequences thereof or a fusion peptide consisting of a polypeptide of formula I or partial sequences thereof and the amino acid sequence of the β-galactosidase in whole or in part, and the gene expressed to form a polypeptide of formula I or a fusion protein consisting of a polypeptide of formula I and the amino acid sequence of the β-galactosidase, in whole or in part, and an resulting fusion protein is cleaved to form a polypeptide of formula I. I I 175337 B1 I I 20 I Method according to claim 1, characterized in that a gene whose non-coding strand hybrid I is integrated with that of the polypeptide of the formula I coding gene I coding strand is incorporated. A method according to claim 1 or 2, characterized in that the gene is chemically synthesized, preferably I by the phosphite method. IN Method according to one or more of the preceding claims, characterized in that the gene in connection I I. 10 to the codon of the amino acid in the carboxy end of the loop · I contains two stop codons. IN Method according to one or more of the preceding claims, characterized in that the gene corresponds to the I I DNA sequence I. I I 6. The DNA sequences I, IA, IB, IC, IIA and IIB. IN A fusion peptide, characterized in that the I I consists of a polypeptide according to claim 1 and the amino acid sequence of the β-galactosidase I whole or in part. II S. Hybrid plasmid, characterized in that it II 20 contains a DNA sequence encoding the polypeptide of II formula I. II 9. Host organism, preferably E. coli, characterized in that it contains a hybrid plasmid of II claim 8. II 25 I 1 I 35 I 30 I