Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1991-05-24
2002-01-29
Yucel, Remy (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...
C435S440000, C435S471000, C435S483000, C435S252300, C435S252330, C435S254110, C435S254210, C435S320100, C530S300000, C530S324000, C530S350000
Reexamination Certificate
active
06342373
ABSTRACT:
The invention relates to DNA sequences which code protease inhibitors designated eglins, hybrid vectors containing such DNA sequences, hosts transformed by such hybrid vectors, novel potypeptides which have protease inhibitor activity and have been produced by such transformed hosts, processes for the preparation of these DNA sequences, hybrid vectors and transformed hosts, and processes for the preparation of eglins with the aid of the transformed microorganisms.
Two protease inhibitors which are isolated from leeches (
Hirudo medicinalis
) and which are designated eglin B and eglin C are known from German Offenlegungsschrift 2,808,396. These polypeptides each consist of 70 aminoacids, have a molecular weight of about 8,100 and are potent inhibitors for chymotrypsin, subtilisin, the animal and human granulocyte proteases elastase and cathepsin G and the mast cell protease chymase (1). Trypsin-like proteases are inhibited to a lesser degree.
Eglin C has the following primary structure (2):
ThrGluPheGlySerGluLeuLysSerPheProGluValValGlyLysThrVal AspGlnAlaArgGluTyrPheThrLeuHisTyrProGlnTyrAspValTyrPheLeuProGluGly SerProValThrLeuAspLeuArgTyrAsnArgValArgValPheTyrAsnProGlyThrAsnVal ValAsnHisValProHisValGly
In contrast to most of the known proteinase inhibitors, eglin C contains no disulfide bridge and, even for a miniprotein, it proves to be unusually stable towards denaturation by acid, alkali or heat and towards proteolytic degradation. The primary structure of eglin B differs from that of eglin C by replacement of the aminoacid 35, tyrosine, by histidine.
The eglins belong to the most potent inhibitors known at present for human and animal granulocyte elastase, and for human granulocyte cathepsin G and bacterial proteases of the subtilisin type. Uncontrolled or excessive release of these cellular proteases in the organism can intensify an inflammation process and cause tissue degradation by non-specific proteolysis. This is particularly due to the fact that these enzymes, which are responsible for intracellular digestion, have an optimum action in the physiological (neutral to weakly alkaline) medium and are capable of rapidly destroying and inactivating natural tissue substances (for example elastin) and humoral factors (for example blood coagulation factors and complement factors). On the basis of their properties known so far, the eglins are therefore of great interest for use in medical therapy (antiinflammation, antiphlogistics, septic shock, pulmonary emphysema, mucoviscidosis and the like).
Only very small amounts of eglins are formed in leeches (about 16 &mgr;g/leech). Isolation and purification of the eglins from leeches is therefore very time-consuming and expensive and cannot be carried out on a commercial scale.
On the basis of the enormous advances in so-called recombinant DNA technology (or genetic engineering), it has recently become possible to prepare the most diverse physiologically active polypeptides using this technology.
The present invention is based on the object of providing, with the aid of genetic engineering means, expression systems which allow the microbial preparation of eglins on an industrial scale. In the present invention, this object is achieved by providing hybrid vectors containing a DNA sequence which codes an eglin and which is regulated by an expression control sequence such that an eglin is expressed in a host transformed by these hybrid vectors.
Preparation of DNA Sequences which Code an Eglin
The invention relates to DNA sequences which code an eglin, for example eglin B and, in particular, eglin C, or a modified eglin, for example modified eglin B or, in particular, modified eglin C, the modification consisting of a shortening of the primary structure of the eglin whilst maintaining the eglin activity, and fragments thereof.
Unless defined more specifically, the general designation “eglins” in the context of the present invention is to be understood as meaning polypeptides with proteinase inhibitor activity, the primary structure of which largely corresponds to the primary structures of eglin B or C (structure homology in general up to 80%), but which can also be modified N-terminally, for example N&agr;-acetylated, N
&agr;
-methionylated or N
&agr;
-acetylmethionylated on the threonine.
In the case of modified eglins, the modification preferably consists of a shortening of the primary structure of the natural eglins, for example by 1 to 10, in particular 1 to 6, aminoacid units at the N-terminus and/or by 1 to 6, in particular 2, aminoacid units at the C-terminus, derivatives modified on the N-terminus, for example acetylated and methionylated or N-acetylmethionylated derivatives, also being included here.
The invention furthermore relates to processes for the preparation of DNA sequences which code an eglin, for example eglin B and, in particular, eglin C, or a modified eglin, for example modified eglin B or, in particular, modified eglin C, and of fragments thereof, which comprises isolating the eglin structure geno from genomic leech-DNA, or preparing a complementary double-stranded eglin-DNA (eglin-ds cDNA) from eglin-mRNA, and, for the preparation of DNA sequences which code a modified eglin, treating the genomic eglin structure gene or the eglin-ds cDNA with suitable nucleases, or which comprises preparing a corresponding (modified) eglin structure gene or fragments thereof by means of chemical and enzymatic processes.
Genomic eglin-DNA and eglin-ds cDNA are obtained, for example, by methods which are known per se. Thus, genomic eglin-DNA is obtained, for example, from a leech gene bank containing the eglin gene, by cloning the leech-DNA fragments in a microorganism and identifying clones containing the eglin-DNA, for example by colony hybridisation using a radioactively labelled eglin-DNA-specific oligodeoxynucleotide containing at least 15, preferably 15 to 30, deoxynucleotides. The DNA fragments thus obtained as a rule contain, in addition to the eglin gene, further undesired DNA constituents, which can be detached by treatment with suitable exo- or endonucleases.
Double-stranded eglin-cDNA can be prepared, for example, by obtaining mRNA from suitable leech cells, preferably those which have been induced into eglin formation, enriching the eglin-mRNA in the resulting mRNA mixture in a manner which is known per se, using the mRNA as a template for the preparation of single-stranded cDNA, synthesising the ds cDNA therefrom with the aid of an RNA-dependent DNA-polymerase and cloning this in a suitable vector. Clones containing the eglin-cDNA are identified, for example, as described above, by colony hybridisation using a radioactively labelled eglin-DNA-specific oligodeoxynucleotide.
To prepare DNA sequences which code modified eglins, the genomic eglin-DNA or eglin-cDNA obtainable can be treated with suitable exo- and/or endo-nucteases which detach the DNA sections coding the N- or C-terminal eglin aminoacids.
The genomic eglin-DNA obtained in this manner or the eglin-cDNA are preferably linked on the 5′- and on the 3′-end with chemically synthesised adapter oligodeoxynucleotides which contain the recognition sequence for one or more restriction endonuclease(s) and thus facilitate the incorporation into suitable vectors. In addition, the adapter molecule for the 5′-end of the eglin-DNA or -cDNA must also contain the translation start signat (ATG). The translation start signal must be located such that it is followed directly by the codon for the first aminoacid of the eglin.
Since the structure of the natural eglin gene is unknown and the chemical synthesis of an eglin gene offers advantages, especially in respect of time, on the basis of modern synthesis possibilities, chemical synthesis is a preferred embodiment of the present invention.
Chemical Synthesis of an Eglin Gene
The invention particularly relates to a process for the preparation of a structure gene for an eglin or for a modified eglin or of fragments thereof, which comprises chemically synthesising segments of the coding and complementary strand o
Alkan Sefik
Fritz Hans
Liersch Manfred
Märki Walter
Meyer Francois
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
UCP GEN-Pharma AG
Yucel Remy
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