Secretion of hirudin derivatives

Details Secretion of hirudin derivatives Secretion of hirudin derivatives

1999-04-02

2003-02-04

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C514S012200, C530S324000

Reexamination Certificate

active

06514730

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for obtaining hirudin derivatives from
E. coli
secretor mutants, and to a hirudin derivative with the N-terminal amino-acid sequence (SEQ ID NO: 1).
2. The Prior Art
Hirudin is a polypeptide with 65 amino acids and was originally isolated from the leach
Hirudo medicinalis
. It acts as a highly specific inhibitor of thrombin by forming stable complexes with thrombin and, therefore, has many possible therapeutic uses, especially for anticoagulation therapy (F. Markquardt, Biomed. Biochim. Acta 44 (1985), 1007-1013).
The publication of the complete amino-acid sequence of hirudin (J. Dodt et al., FEBS LETTERS 165 (2), (1984), 180-184) was the prerequisite for the preparation of hirudin by recombinant DNA techniques and expression in microorganisms.
European Patent Application No. 158,564 (Transgene) discloses cloning vectors for the expression of hirudin or hirudin analogues in a host cell, especially a bacterial cell. The gene coding for hirudin is, in this case, obtained by cDNA synthesis starting from mRNA from the leach
Hirudo medicinalis
. Described, in particular, is a hirudin derivative with the N-terminal sequence (SEQ ID NO: 2) and processes for obtaining it.
European Patent Application No. 168,342 (Ciba Geigy) discloses DNA sequences which code for the natural amino-acid sequence of hirudin, wherein the N-terminal amino-acid sequence is (SEQ ID NO: 3). The expression of hirudin takes place intracellularly in the microorganisms
E. coli
and
Saccharomyces cerevisiae.
European Patent Application No. 171,024 (Hoechst AG) discloses a process for the genetic engineering for preparation of polypeptides with hirudin activity, in particular, in
E. coli
cells, wherein the cells are disrupted and the polypeptide with hirudin activity is obtained from the cell extract. A fusion protein portion which is present where appropriate can be deleted by proteolytic or chemical cleavage, and the liberated hirudin molecule can be purified.
German Patent Application No. 3,445,571 (GEN-BIO-TEC) relates to a DNA sequence which codes for a protein with the biological activity of hirudin, and to a process for obtaining such proteins from
E. coli
cells which are transformed with a suitable recombinant vector by lysis of the cells.
The paper by Bergmann et al (Biol. Chem. Hoppe Seyler 367 (1986), 731-740) also describes hirudin synthesis in
E. coli
. The hirudin is released from the cells by toluene treatment, with only low yields of about 500 ng/l A
578
units of cells being achieved.
European Patent Application No. 200,655 (Transgene), European Patent Application No. 252,854 (Transgene), and European Patent Application No. 225,633 (Ciba Geigy) disclose the obtaining by secretion of proteins with hirudin activity from a eukaryotic host organism, especially yeast, wherein the expression takes place on a vector which contains a DNA sequence which contains a signal peptide upstream of the structural gene. The secretion of hirudin derivatives with the N-terminal sequence (SEQ ID NO: 3) and with the N-terminal sequence (SEQ ID NO: 2) in yeast is disclosed. In this case, yields of up to 100 mg/l are reported.
German Patent Application No. 3,900,626 (Hoechst AG) discloses a hirudin derivative with the N-terminal sequence (SEQ ID NO: 4). The expression takes place preferably in yeast, using the promoter and signal sequence of the yeast pheromone gene MF&agr; for the expression and secretion of the hirudin derivative.
All the processes described above for preparing hirudin derivatives have disadvantages, however. Thus, when yeast is used as the host organism, and the hirudin is secreted into the culture medium, relatively high yields are obtained, but the cultivation of yeast cells takes longer and is more demanding than that of bacteria, for example,
E. coli
. However, on the other hand, in
E. coli
cells, the yield is relatively low, and/or complicated isolation processes are necessary on disruption of the cells.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to develop a straightforward process for obtaining hirudin derivatives in which hirudin derivatives can be obtained in high yield from bacterial cells without entailing the necessity of disruption of the cells.
The present invention relates to a process for obtaining hirudin derivatives from
E. coli
secretor mutants which entails:
(1) construction of a recombinant vector on which there Is located the gene coding for a hirudin derivative downstream of a DNA section which codes for a bacterial signal peptide;
(2) transforming an
E. coli
secretor mutant with the recombinant vector constructed in step (1);
(3) cultivating the transformed cells in a medium; and
(4) obtaining the hirudin derivative from the medium.
The term “hirudin derivative,” according to the present invention, refers to proteins which are derived from hirudin which act as thrombin inhibitors and have a specific activity of at least 10,000 AT-U/mg (antithrombin units) (Dodt et al., Biol. Chem. Hoppe Seyler 366 (1985), 379-385). The term “hirudin derivative” also comprises fusion proteins with an N-terminal fusion portion which is up to about 50 amino acids long and can be partially or completely deleted by proteolytic or chemical cleavage, resulting in, as a cleavage product, a hirudin derivative of a specific activity of at least 10,000 AT-U/mg.
Preferably obtained by the process according to the invention are hirudin derivatives with the following N-terminal amino-acid sequence:
(X)
m
—Z—  (SEQ ID NO: 5)
in which
m
0 to 50;
x
represents identical or different genetically
encodable amino acids;
z
represents a genetically encodable amino acid from
the group comprising Leu, Ile, Ala, Val, Gly, Ser,
Asp, Glu, Asn, Gln, His, Met, Phe and Tyr.
Where m is greater than 0, the sequence X preferably contains a proteolytic or chemical cleavage site, particularly preferably at its end. If, for example, the last amino acid in the sequence X is an Arg residue, the fusion sequence X can be cleaved off by digestion-with trypsin (cleavage after Arg), and the active hirudin derivative can be purified. However, it is equally possible to cleave off the fusion portion using other known proteolytic enzymes or chemical cleavage reagents. If, for example, the amino-acid sequence of X terminates with a Met residue, the fusion protein can be cleaved by cleavage with cyanogen halides (E. Gross and B. Wittkop, J. Am. Chem. Soc. 82 (1961) 1510-1517). If, for example, the C-terminal amino-acid sequence of X contains the amino-acid sequence (SEQ ID NO: 6), the cleavage can be carried out with factor Xa (European Patent Application No. 25,190 and European Patent Application No. 161,973).
When m=0, in the process according to the invention, Z preferably represents Ala, Gln, His, Phe, Tyr, Gly, Ser, Asp or Asn, particularly preferably Ala, Gly, Ser, Asp or Asn. Maximum preference is given to a hirudin derivative in which m denotes 0 and Z represents Ala.
Thus, the present invention also relates to hirudin derivatives with the N-terminal sequence A—(SEQ ID NO: 5) in which A represents Ala, Gln, His, Phe, Tyr, Gly, Ser, Asp or Asn, preferably Ala, Gly, Ser, Asp or Asn. Maximum preference is given to a derivative with the N-terminal sequence (SEQ ID NO: 1). Surprisingly, it has been possible to obtain from this hirudin derivative in the culture supernatant of an
E. coli
secretor mutant up to above 2 g/l medium of active hirudin.
Another advantage of the process according to the invention is that, owing to the secretion of the hirudin derivative into the cell medium, the disulfide linkages of hirudin are correctly formed under the oxidative conditions of the medium.
According to the present invention, the term
E. coli
secretor mutants is intended to refer to
E. coli
strains which show massive protein secretion into the culture medium. A process for preparing these secretor mutants is disclosed in European Patent No. 338,410. The obtaining of suitable
E.

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