Ancrod proteins, their preparation and use

Details Ancrod proteins, their preparation and use Ancrod proteins, their preparation and use

1991-05-14

2000-01-18

LeGuyader, John L.

Chemistry: molecular biology and microbiology

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

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

4351723, 4352523, 43525233, 4352542, 4353201, 435325, 536 231, 536 235, C12N 1512, C07H 2104

Patent

active

060156856

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

The present invention relates to ancrod proteins, their preparation and use for the prophylaxis and therapy of diseases.
Ancrod is a fibrinogen-splitting enzyme which can be obtained from the venom of the Malayan pit viper (Agkistrodon rhodostoma) and has anticoagulant properties (Biochem, J. 131 (1973) 799).
Ancrod has a molecular weight of about 38,000 Dalton and a carbohydrate content of about 38%.
The process for preparing ancrod is elaborate. The extremely venomous Malayan pit viper must be raised in snake farms and milked by hand before the biochemical processing of the secretion for the isolation of ancrod can be started. Ancrod prepared in this way can be used for only a limited time because signs of resistance may appear after 6 to 8 weeks and are presumably due to the formation of ancrod-neutralizing antibodies. In isolated cases there are also hemorrhagic complications.
We have now found and prepared pure ancrod proteins which are superior, in terms of therapeutic use and the preparation, to the ancrod obtainable hitherto.


SUMMARY OF THE INVENTION

The present invention relates to pure glycosylated, partially glycosylated or unglycosylated polypeptides having the following aminoacid sequence:


1 VIGGDECNIN EHRFLVAVYE GTX.sup.1 WTFICGG VLIHPEWVIT AEHCARRRMN - 51 LVFGMHRKSE KFDDEQERYP KKRYFIRCX.sup.2 K TRTSWDEDIM LIRLNKPVX.sup.3 N - 101 SEHIAPLSLP SNPPIVGSDC RVMGWGSINR RIHVLSDEPR CANINLHX.sup.4 FT - 151 MCHGLFRKMP KKGRVLCAGD LRGRRDSCNS DSGGPLICNE ELHGIVARGP - 201 NPCAQPNKPA LYTSVYDYRD WVNNVIAGX.sup.5 A TCSP natural .alpha.-aminoacids.
The individual letters therein denote the aminoacids (cf. Lubert Stryer, Biochemie, 1979, p. 12, S. R. Vieweg).
The amino-acid residues X.sup.1, X.sup.2, X.sup.3, X.sup.4 and X.sup.5, which can be identical or different, represent N, Q, S, T, G, D, E, K, R, P, but preferably N, Q, S and T and, in particular, N and Q.
The present invention also relates to DNA sequences which code for the abovementioned proteins, as well as to vectors which contain these DNA sequences.
The proteins according to the invention can be prepared by known methods of genetic manipulation.
Thus, it is possible for mRNA to be isolated from the glandular tissue of a Malayan pit viper (Agkistrodon rhodostoma) and to be converted into double-stranded cDNA. After this cDNA has been inserted into a commercial cloning vector, for example .lambda. gt 10, a cDNA library is set up. For the methods used for this, reference may be made, for example, to Maniatis et al., Molecular Cloning, CSH Press (1982). The screening of such gene banks with radiolabeled oligonucleotide probes is by now also a widely used and described method. It is possible by this process to isolate and characterize a cDNA clone which has homology with the oligonucleotide probe. This process is described in "DNA cloning Vol. I, IRL Press, 1985.
It is easy with the aid of restriction enzymes to obtain the cDNA characterized in this way. The fragments obtained thereby can be used, where appropriate in conjunction with chemically synthesized oligonucleotides, adaptors or gene fragments, to clone the sequences coding for the protein. The incorporation of the gene fragments or synthetic DNA sequences into cloning vectors, for example the commercial plasmids M13mp or pkk-223-3, is carried out in a conventional manner. It is also possible to provide the genes or gene fragments with suitable control regions which have been chemically synthesized or isolated from bacteria, phages, eukaryotic cells or viruses thereof and which make it possible for the proteins to be expressed.
The transformation or transfection of suitable host organisms with the hybrid plasmids obtained in this way is likewise known and described in detail (M. Wigler et al., Cell 16 (1979) 777-785; F. L. Graham and A. J. van der Eb, Virology 52 (1973) 456-467). It is also possible to provide the hybrid plasmids with appropriate signal sequences which allow the polypeptides to be secreted into the medium.
The vectors which can

REFERENCES:
patent: 3879369 (1975-04-01), Nolan
patent: 4154656 (1979-05-01), Maurer
Studies on the Coagulant Enzyme from Agkistrodon Rhodostoma Venom, Hatton, Biochem. J. (1973) 799-807, vol. 131.
Characterization of a Protein C Activator from Agkistrodon Contortrix Contorix Venom, Kisiel et al., The Journal of Biological Chemistry, vol. 262, No. 26, Sep. 15, 1987 12607-12613.
Primary Structure of a Protein C Activator from Agkistrodon Contorix . . . Biochemistry 1989, 28, 674-679, McMullen et al.

Affiliated with

Also associated with

Rating

Ancrod proteins, their preparation and use does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Ancrod proteins, their preparation and use, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Ancrod proteins, their preparation and use will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-562255