Fibronolytically active polypeptide

Details Fibronolytically active polypeptide Fibronolytically active polypeptide

1999-10-01

2001-07-17

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S219000, C424S094670

Reexamination Certificate

active

06261820

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a fibrinolytically active metalloproteinase of non-naturally occurring sequence, to combinant methods for its manufacture, and to its use in treating thrombosis in vivo.
BACKGROUND OF THE INVENTION
Fibrolase is an enzymatically active polypeptide (specifically, a metalloproteinase) composed of 203 amino acid residues that was originally isolated by purification from the venom of the Southern Copperhead snake; U.S. Pat. No. 4,610,879, issued Sep. 9, 1986 (Markland et al.); and Guan et al., Archives of Biochemistry and Biophysics, Volume 289, Number 2, pages 197-207 (1991). The enzyme exhibits direct fibrinolytic activity with little or no hemorrhagic activity, and it dissolves blood clots made either from fibrinogen or from whole blood.
The amino acid sequence of fibrolase has also been determined, with methods described for recombinant production in yeast and use for the treatment of thrombembolic conditions in vivo; Randolph et al., Protein Science, Cambridge University Press (1992), pages 590-600, and European Patent Application No. 0 323 722 (Valenzuela et al.), published Jul. 12, 1989.
SUMMARY OF THE INVENTION
This invention provides a fibinolytic metalloproteinase having the non-naturally occurring linear array of amino acids depicted in SEQ ID NO: 1, also referred to herein as “novel acting thrombolytic” (or “NAT”). Also provided are nucleic acid molecules, such as the one of SEQ ID NO: 2 and variants thereof encoding NAT.
The term “mature” is used in its conventional sense to refer to the biologically active polypeptide which has been enzymatically processed in situ in the host cell to cleave it from the prepro region.
Because of its fibrinolytic activity, NAT is useful in vivo as a blood clot lysing agent to treat thrombosis in a mammal (including rats, pigs and humans).
The NAT polypeptide of this invention provides advantages over naturally occurring fibrolase as a therapeutic agent (i.e., the fibrinolytic polypeptide found in snake venom). Native fibrolase is known to contain several alternate N-termini: QQRFP (amino acids 1-5 of SEQ ID NO: 5), EQRFP (amino acids 1-5 of SEQ ID NO: 15) and ERFP (amino acids 1-4 of SEQ ID NO: 16) (in which “E” designates a cyclized glutamine, or pyroglutamic acid). More specifically, starting with an N-terminus composed of QQRFP (see SEQ ID NO: 5), the fibrolase molecule undergoes degradation to result in two isoforms, having N-terminal sequences of EQRFP (see SEQ ID NO: 15) and ERFP (see SEQ ID NO: 16), respectively. Recombinant fibrolase as produced in yeast typically yields a mixture of all three of these forms and is thus not homogeneous. See Loayza et al., Journal of Chromatography, B 662, pages 227-243 (1994). Moreover, the cyclized glutamine residue results in a “blocked” N-terminus which makes sequencing impossible.
In contrast, the recombinant NAT of this invention provides a single species: only one N-terminus is typically produced. The result is greater homogeneity of the end product compared to recombinant fibrolase, which is beneficial when medical applications are the intended end use.


REFERENCES:
patent: 4610879 (1986-09-01), Markland et al.
patent: 0 323 722 (1989-07-01), None
Guan et al., Archives of Biochemistry and Biophysics, vol. 289, No. 2, pp. 197-207 (1991).
Randolph et al., Protein Science, , vol. 1, pp. 590-600 (1992).
Markland et al., Circulation, vol. 90, No. 5, pp. 2448-2456 (1994).
Loayza et al., Journal of Chromatography B 662, pp. 227-243 (19940.
Rholam et al., European Journal of Biochemistry, vol. 227, pp. 707-714 (1995).

Affiliated with

Also associated with

Rating

Fibronolytically active polypeptide does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Fibronolytically active polypeptide, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Fibronolytically active polypeptide will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2452868