CA2066442A1

CA2066442A1 - Method of preparation of hirudin

Info

Publication number: CA2066442A1
Application number: CA002066442A
Authority: CA
Inventors: Wolfgang Koerwer
Original assignee: Individual
Current assignee: BASF SE
Priority date: 1989-12-22
Filing date: 1990-12-04
Publication date: 1991-06-23
Also published as: DE59006998D1; EP0506833B1; ATE110775T1; EP0506833A1; DE3942580A1; WO1991009946A1; JPH05502672A

Abstract

Described is the use of fusion proteins comprising protein A and hirudin peptides to prepare hirudin peptides.

Description

~J~ 3~
O.Z. 0050/41318 The preparation of hirudin Description The present invention relate~ to a proce~s for the preparation of hirudin with the aid of a novel fu~ion prot~in.
Hirudin ha~ been known for a long tims ~cf. Merck Index 1983, No. 4613~ and ha~ anticoagulant propertie~.
The structure of hirudin is likewise known (FEBS Lett.
165 ~1984) 180). Hirudin may be ~ulfa~ed on the amino acid tyrosine in position 63.
There have been attempts to prepare hirudin by gene manipulation (FEBS Lett. 202 (1986) 373, Biol. Chem.
Hoppe-Seyler 367 (1986) 731). ~owever, the yields were poor because hirudin was evidently unstable to proteo-lysis in the cell or expression was very low. Although expression as a fusion protein produced higher crude yields, in this case purification is very elaborate (DNA 5 (1986) 511).
We have now found that hirudin peptides can be prepared with good yields and straightforward purifica-tion via a fusion protein of the formula X-Y-hirudin peptide where X is protein A or one of its active polypeptide fragmentsJ and Y i8 Met or an oligopeptide sequence which can be cleaved at the point of attachment to the hirudin or in the neighborhood thereof.
The term hirudin peptide includes hirudin, hirudin extended or truncated at the N terminus by 1-3 amino acids, and hirudin peptide~ with hirudin activity.
Protein A i~ a known protein tcf. EP 135 532).
The term "active polypeptide fragment" of protein A means thoss protein A fragments which still have the ability to bind to immunoglobulin (cf. Biochemistry 20 (1981) 2361-96 and Eur. J. Biochem. 156 (1986) 637-43).
Y i~ Met or an oligopeptide sequence which i5 ~ ~ ~ 6 ~

- 2 - O.Z. 0~50/41318 present neither in X nor in hirudin and which can be cleaved enzym~tically or chemically at the point of attachment to the hirudin or in the neighborhood thereof.
In the sLmplest case, Y is methionine.
The fusion protein can be cleaved by conventional methods. If Y is methionene [sic], the cleavage can be carried out with cyanogen bromide, in which ca~e X-homo-serine and hirudin are liberated. When Y contains the sequence Asp-Pro, it i~ possible to use acids to cleave between these amino acids. In this case, the Asp residue remains on the protein A carboxyl terminus, and Pro remains on the hirudin N terminus. Other oligopeptide sequences ~uitable for Y ara the following, inter alia:
Asp~Gly (cleavage by hydroxylamine), Pro-Phe-His-Leu~Leu (enzymatic cleavage by renin), Ile-51u-Gly-Arg~hirudin (enzymatic cleavage by factor VIII).
Thus, depending on the oligopeptide Y used, either there is liberation of hirudin or hirlldin peptide or the result is extended hirudin or hirudin peptides.
The hirudin peptide can have the originally published amino acid sequence (see FEBS Lett. 165 (1987 [sic]) 180~ or the sequence of one of the isohirudins which have been found since (FEBS Lett. 255 (1989) 105-110) or of artificially prepared mutants with hirudin activity.
The present invention also relates to the above-mentioned X-Y-hirudin fusion protein and the use ~hereof for the preparation of hirudin.
The fu~ion protein which was produced by replac-ing the 388 bp N-terminal protein A fragment from the commercial vector PRIT 2T r 8iC ] ( Pharmacia~ Order No.
27-4808-01, Fig. 1~ by a 97 bp synthetic adaptor (seguence 1) has proven particularly advantageous. The DNA ~equence and the relevant amino acid sequence are depicted a~ ~equence 7. Amino acids 1-163 are protein ~-peptide X, amino acid~ 164-175 are oligopeptide Y and amino acids 176-230 are the hirudin sequence.

J l3~

- 3 - O.Z. 0050/41318 The fusion proteins have the following excep-tional properties:
1) Very high-level expression is possible in E. coli.
2) They are very stable to proteolysis.
3) They are produced in soluble form in E. coli.

4) They are very stable to heat (up ~o ~ 80C).

5) They bind to IgG-Sepharose exclusively via their protein A portion.
The fusion protein of Example 1 additionally has the following advantages:
a) Cleavage with BrCN produces mature hirudin without an additional N terminal amino acid.
b) Since the methionine in the cleavage peptide [sic]
is the only one in the fusion protein, cleavage produces two fragments. Of these, the fusion partner can be ~eparated from hirudin by IgG affinity chromatography.
~XAMPLE 1 Preparation of the expression plasmid a) Construction of the vector:
The protein A vector pRIT 2T (Eigure 1) is commercially available and has been described in detail (Pharmacia Order No. 27-4808-01).
This vec$ox was modified as follows: it was cleaved with the restriction endonuclease Hind III. The larger fragment (~ector) was isolated from an agarose gel by electroelution. The complementary oligonucleotides Roe 1/2 (sequence 1) were ligated into this vector. The resulting chimeric pla~mid was transformed into the lambda lysogenic strain N 4830-1 (Pharmacia Order No.
27-4808-01 [sic~3. The clone with the correct orientation of the oligonucleotides was found, with the aid of Hind III/EcoRI restriction mapping, from among the possible recombinants, and was checked by DNA sequencing.
This expres~ion plasmid was called pRIT 2TA.
b) Insertion of a synthetic hirudin gene with adaptor The pRIT 2TA DNA wa~ cut with EcoRI and SalI, and - ~ _ o.z. 0050~41318 the larger DNA fragment (a) wa~ isolatsd from an agaro~e gel by electro~lution.
A synthetic hirudin gene (sequence 6) wa~
prepared usin~ a DNA ~yntheslzer (Applied Biosystems, model 380A). 4 Oligonucleotides (Koe 3-Xoe 6; sequencas 2-5) were prepared for this. The oligonucleotides were kinased and ligated to the EcoRI/SalI-linearized plasmid pUC 18. The construct wa~ checked by DNA sequencing. The hirudin gene (b) including tha adaptor was cut out of thi~ chimeric plasmid (pUC 18-Hir) with EcoRI and isolated by agarose gel electrophoresis and electro-Plution. Besid~s two stop codons at the 3' end and the SalI recognition site, the synthetic hirudin gene con-tains an adaptor sequence which links the hirudin gene to the protein A fusion partner via the EcoRI cleavage site, with retention of the reading frame.
The isolated DNA fragments a and b were ligated together and transformed into the lysogenic strain N 4830-1. This resulted in the protein A-hirudin expres-sion vector pRIT2TA-~ir (Fig. 2).

Expression of the fusion protein The expression plasmid pRIT 2T~-Hir was trans-formed into the E. coli strain N 4830-1 (Pharmacia Order No. 27-4808-01 ~sic]). This strain contains the thexmosen~itive lambda repressor CI [sic] 857 in the chromosome.
100 ml of MIM medium (MIM = 32 g of tryptone, 2U g of yea~t extract, 6 g of Na2HPO4, 3 g of RH2PO4, 0.5 g of NaCl, 1 g of NH4Cl per liter and 0.1 mM MgSO4 plus 0.001 m~ FeCl3) were sterilized in a 1 1 Erlenmeyer flask with baffles, and amplcillin waY added (ad 100 ~g/ml).
The medium was inoculated with 1 ml of a fresh overnight culture of the strain pRIT 2TA-Hir/N 4830-1 and incubated at 28C with shaking, until the absorption at 550 nm was 0.6. Then 100 ml of fxesh MIM/amp medium at 65~C were add d, and incubation was continued at 42C for 4 h. The - ~ - O.Z. 0050/41318 desired fusion protein was synthesi~ed during this time.
The cell walls were removed enzymatically by adding lysozyme to 75 mg/l and incubation ~37C, 3 h). It was then possible to disrupt these cell~ mechanically (~anton-Gaulin press, freezing cycle, vigoxous stirring), by a heat shock up to 80C or by hypotonic lysis, to release the soluble fllsion protein into the medium.

Purification of the fusion protein The cell fragments were removed by centrifuga-tion, and the clear supernatant was pumped through an IgG-Sepharose column (IgG Sepharose~ 6 Fast Flow, Pharmacia, Order No. 17-0969-01). The manufacturer's instructions were followed in the storage of the column material, preparation and setting up of the column, application conditions and flow rates. Thus, a 200 ml gel bed and a flow rate o about 3 l~h were used for 6 l of supernatant. In this step the fusion protein was revers-ibly bound via its IgG-binding protein A portion to the gel matrix (yield about 95%). After application, the column was washed with 10 bed volumes of TST (50 mM
tris-HCl, pH 7.6; 150 mM NaCl and O.05~ Tween~20) and eluted with 0.5 M acetat~ buffer, pH 2.8.

Cleavage of the fusion protein The column eluate from Example 3 was lyophilized and stored at -20C. For the cleavage, it was taken up in 70% strength formic acid to a protein concentration of about 25 g/l After flushing with argon, 1 g of solid BrCN was added per g of fusion protein to cleave off the hirudin. The cleavage took place under argon at 37C in about 4 h. The excess cyanogen bromide, the solvent and other volatile components were removed by lyophiliæation.
The material was then washed three tLme~ with wat~r.

Rena~uration and purification of ~he hirudin The lyophilizate was taken up in 6 M guanidinium ` '~ ,'i ~ ,1 1 ;, , " ~ ~ ~3 ~

- 6 - o.æ. 0050/41318 hydrochloride, 0.1 M tris/HCl, p~ a.s, 0.2 ~ DTT to protein concentration of 1-100 mg/ml. The sample wa~
incubated for 2 h and then desalted by G-10 exclusion chromatography (equilibrated with 10 M HCl). The desalted sample was diluted 1:20 in 0.1 M tris/HCl, 5 mM GSH/
0.5 mM GSSG, 1 mM EDTA, pH 8.7 and incubated for 1 h (GSH
is reduced and GSSH [sic] is oxidized glutathiane). This treatment increased the specific activity of the hirudin by a factor of 3-5. The pH was adjusted to 7.6 with HCl, 10 NaCl was added to 150 mM and Tween~20 was added to 0.05~, and then the IgG-Sepharose chromatography was repeated (Example 3). While the protein A fusion partner and uncleaved fusion protein were bound to the column, the active hirudin was present with a purity ~ 90% in the 15 flow-through. Classical methods of protein chemistry could be used to purify it to clinical purity.

Sequences Sequence l:

Hind 111 Ser Asn ASn Phe Asn Lys Glu Gln Gln Asn Ala Phe Koe 1 5 -A GCT TCT AAC AAT TTC AAC AAA GAA CAA CAA AAT GCT TTC
Koe 2 3 -AGA TTG TTA AAG TTG TTT CTT GTT GTT TTA CGA AAG

Tyr Glu lle Leu ~is Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly TAT GAA ATC TTG CAT TTA CCT AAC TTA AAC GAA GAA CAA CGC AAT GGT
ATA CTT TAG AAC GTA AAT GGA TTG AAT TTG CTT CTT GTT GCG TTA CCA
Hind III
Phe lle Gln Sequence 2 ~oe 3: 5 -AATTCAAAAA CCAAACCGCG TATCAAAACC ATGGTTGTTT ACACTGACTG CACTGAATCC
GGTCAGAACC TGTGCCTGTG CGAAGGCTCT AACGTTTGCG GCCAGGGCAA CAAATGCATC

5 ~ ",

- 7 - O. Z . 0050/4131 Sequence 3 o~ 4: 5 ~GACGGCGAAA AAAACCAGTG CGTTACTGGC GAAGGTACCC CGAAACCGCA GTCTCACAAC

Se~uence 4 oe 5: 5 -TCGACCTATT ACTGCAGGTA TTCTTCCGGG ATTTCTTCGA AGTCGCCGTC GTTGTGAGAC
rGCGGTTTCG GGGTACCTTC GCCAGTAACG CACTGGTTTT TTTCGCCGTC AGAGCCCAGG

Sequence 5 oe 6: 5 -GTTGCCCTGG CCGCAAACGT TAGAGCCTTC GCACAGGCAC AGGTTCTGAC CGGATTCAGT

Saquence Eco Rl GGTCAGAACC TGTGCCTGTG CGAAGGCTCT AACGTTTGCG GCCAGGGCAA CAAATGCATC
CCAGTCTTGG ACACGGACAC GCTTCCGAGA TTGCAAACGC CGGTCCCGTT GTTTACGTAG
CTGGGCTCTG ACGGCGAAAk AAACCAGTGC GTTACTGGCG AAGGTACCCC GAAACCGCAG
GACCCGAGAC TGCCGCTTTT TTTGGTCACG CAATGACCGC TTCCATGGGG CTTTGGCGTC
Sall Sequence 7 ;~el Glu Gln Ar~ Ile Thr Leu Lys Glu Ala Ser Asn Asn Phe Asn Lys ATG GAA CM C(iC ATA ACC CTG AM GM GCT TCT AAC AAT TTC AAC AAA 48 TAC CTT GTT GC(i TAr TliG GAC TTT CTT CGA AGA TT(i TTA AAG TT~ TTT 4 2~ 30 ~il u Gl n Gl n Asn Al a Ph~ T~yr Gl u Il e Leu ~li s Leu Pro Asn Leu Asn GM CM CAA AAT Gcr TTC TAT GAA ATC TTG CAT TTA CCT AAC TTA AAC ~6 CTT ~TT GTT TTA (;GA MG AT~ CTT r~G MC ~TA MT ~GA TTli MT TT~ ~6 .

~'J ~ 3 . ~,~

- 8 - O.Z. 0050/41318 Sequence 7 4~ ~S
~lu ~lu ~ln Ary Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser ~AA GAA CAA C~C AAr G~T TTC ATC CAG AGC TTA AAA GAT GAC CCA A~C 144 CTT crr ~TT ~CG TTA CCA M G TAG UTC ~C~ AAT TTT crA crG GGT TCG 144 5~ 55 ~
Glu Ser Ala Asn Leu Leu Ser Glu ~l d Lys Lys Leu Asn Glu Ser Gln C~A AGT GCT AAC CTA TTG TCA GAA GCT AAA AAG TTA AAT GAA TCT CAA 192 GTT TCA C~ TTG GAT AAC AGT CTT CGA TTT TTC M T TTA CTT AGA GTT 1~2 o~ 7U 75 ~
Ala Pro Lys Al d Asp Asn L~s Pne Asn Lys Glu Gln Gln Asn Al~ Phe GCA CCG AAA GCG GAT AAC ~AA TTC ~AC AAA GAA CAA CAA AAT GCT rTC 240 CGT GGC TTT CGC CTA TrG rTT MG TTG TTT CTT GTT ~TT TTA CGA MG 24

9~ 95 Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Ary Asn Gly ATA CTT TAG AAT ~rA AAT GGA TTG AAT TrG CTT CrT GTT GCG TTA CCA 288

10~ 105 110 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser '~ln Ser Ala Asn Leu Leu TTC ATc C M AGC CTA MA GAT GAC CCA AGC C M AGC GCT AAC CTT TTA 336 AA~ TAG GTT rc~ GAT TTT CrA CTG GGT TC~ ~TT TCG CGA TT~ GM AAT 336

11~ 120 125 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Al d Pro Lys Ala Asp Asn GCA GAA GCT AAA M G CTA MT GAT ~CT CAA GCA CCA A M GCT GAC AAC 3~4 C~T CTT CGA TTT TTC GAT TTA CTA C~A GTT CGT GGT TTr C~A CTG TT~ 3~4 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu TTT AAG TT~ TTT CTT ~TT GTT TTA CGA MG ATA CTT TAA M T GTA M T 432 145 150 155 16~
Pro Asn Leu Thr Glu Glu Gln Ar~ Asn Gly Phe Ile Gln Ser Leu Lys CCT AAC TTA ACT ~M GM CM CGT AAC GGC TTC ATC CAA AGC CTT A M 480 GGA TT~ AAT TGA CTT CTT GTT ~CA TTG CCG AAG TAG GTT TCG GM TTT 48 Asp Asp ~ro Gly Asn Ser Lys Thr Lys Pro Ary Ile Lys Thr '~et ~al GAC GAT CCG GGG AAT TCA AAA ACC AAA CCG CGT ArC AAA ACC ArG GTT 528 CTG CTA ~GC CCC TTA AGT rTT TGG TTT GGC GCA TAG rTT TGG TAC CAA 528 1~ 185 190 Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu GTT TAC ACT ~AC T~C ACT ~AA TCC GGT CAG M C CT~ r~c crG TGC GAA 576 CM ATG TGA CTG ACG TGA CTT AG~ CCA GTC rTG GAC ACG GAC ACG CTT 576 - 9 - O . ~ . 0050/41318 Sequence 7 1~5 l jO 1~5 ~ly Ser Asn Vdl Cys Gly ~iln Gly Asn Lys Cys lle Leu Gly Ser Asp GGC TCT AAC GrT T(iC GGC CAG GGC AAC MA r~c ATC CTG GGC TCT GAC 624 CCG AGA TTG CM ACG CCG Grc CCG Tr(i rTT AC[i TAG GA(: CCG AGA crG 624 20~ 205 ;~1() Gly Glu LJs Asn Gln Cys ~dl Thr Gly Glu Gly Thr Pro Lys Pro Gln GGC GAA AAA MC CA& rGc GTT ACT tiGC GAA GGT ACC CCG AAA CCG CAG 672 CC(i CTT TTT rTG Grc ACG CAA TGA CCG CTT CCA TGG GGC rTT GGC GTC 672 Ser His Asn Asp Gly Asp Phe Glu Glu lle Pro Glu Glu Tyr Leu Gln TCT CAC MC GAC GGC GAC TTC liAA GM Arc CCG GAA GAA TAC CT(i CAG 720 AGA ~T~ TrG crG CCG CTG AAG (;rT CrT TAG GGC CTT CrT ATG GAC GTC 720 Stop Stop ATT ArC 726

Claims

We claim:

1. A process for the preparation of a hirudin peptide, which comprises cleaving a fusion protein of the formula X-Y-hirudin peptide, where X is protein A or one of its active polypeptide fragments, and Y is Met or an oligopeptide sequence which can be cleaved at the point of attachment to the hirudin peptide or in the neighborhood thereof, between protein A and the hirudin peptide.

2. A fusion protein as claimed in claim 1.

3. The use of the fusion protein as claimed in claim 1 for the preparation of hirudin peptides.