Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-09-13
1999-09-28
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435212, 435219, 435226, 4352523, 43525233, 435814, 435815, 530350, 536 231, 536 236, C12P 2106, C12N 950, C07K 14415, C07H 2104
Patent
active
059587229
DESCRIPTION:
BRIEF SUMMARY
The invention concerns an improved process for purifying serine proteases using a recombinant inhibitor DE-3 from Erythrina caffra. Immobilized trypsin inhibitors from Erythrina (ETI) are effective reagents for the affinity chromatographic purification of serine proteases and in particular of plasminogen activators (C. Heussen, J. Biol. Chem. 259 (1984) 11635-11638), .beta.-trypsin, .alpha.-chymotrypsin and thrombin (S. Onesti et al., J. Mol. Recogn. 5 (1992) 105-114). These trypsin inhibitors have been known for a long time (C. Heussen, Haemostasis 11 (1982) P47 (Supplement); F. J. Joubert, Phytochemistry 21 (1982) 1213-1217; F. J. Joubert, Int. J. Biochem. 14 (1982) 187-193).
The inhibitor DE-3 from E. caffra is particularly suitable for the purification of plasminogen activators (F. J. Joubert in Thrombosis and Haemostasis 57 (1987) 356-360). The complete amino acid sequence of this inhibitor is also described in this publication. DE-3 can be isolated and purified from the seeds of E. caffra (F. J. Joubert, Int. J. Biochem. 14 (1982) 187-193).
A recombinant ETI is described by Teixeira et al., Biochimica et Biophysica Acta 1217 (1994) 16-22, whose specific inhibitory activity for tissue plasminogen activator is 1.7.times.10.sup.9 U/mmol. In contrast the specific inhibitory activity of natural ETI is 1.94.times.10.sup.9 U/mmol.
A similar situation applies to the inhibitory activity towards trypsin (2.63.times.10.sup.12 /3.21.times.10.sup.12). Thus the specific inhibitory activity towards trypsin and tissue plasminogen activator of recombinant ETI prepared according to Teixeira is 20% less than the activity of natural ETI.
Recombinant ETI is obtained by expression according to Teixeira and purified by ammonium sulfate precipitation (80% saturation), dialysis against water and a cyanogen bromide cleavage in which the N-terminal sequence including the methionine is cleaved off. It is subsequently chromatographed on an affinity chromatography column (Sephadex G50).
The object of the invention is to improve the effectiveness of processes for the purification of serine proteases using ETI.
The invention concerns a process for the purification of serine proteases from a protein mixture by binding the serine protease to an immobilized polypeptide which has the activity of an inhibitor DE-3 from Erythrina caffra, removing the unbound components from the protein mixture, detaching the serine protease from the inhibitor, separating the immobilized inhibitor from the soluble serine protease and isolating the serine protease which is characterized in that a polypeptide is used as the polypeptide which is the product of a prokaryotic or eukaryotic expression of an exogenous nucleic acid (preferably DNA) and is purified chromatographically by means of an anion exchanger, cation exchanger or a nickel chelate column. surprisingly it was found that the recombinant polypeptide produced according to the invention which has the activity of an inhibitor DE-3 from Erythrina caffra has a substantially increased specific affinity towards serine proteases compared to inhibitor DE-3 from E. caffra isolated from natural sources.
The "activity" of an inhibitor DE-3 from E. caffra is essentially to be understood as its specific inhibitory activity towards serine proteases in particular to tissue plasminogen activators. In this case the specific inhibitory activity of the inhibitor is at 1.07 U/mg or more with regard to trypsin. The inhibition is achieved by binding between inhibitor and serine protease.
The process according to the invention is particularly advantageous for purifying plasminogen activators such as tissue plasminogen activators (t-PA) and derivatives (e.g. mutations and deletions) thereof. t-PA and derivatives are described for example in EP-B 0 093 619, U.S. Pat. No. 5,223,256, WO 90/09437 and T. J. R. Harris, Protein Engineering 1 (1987) 449-458.
The production of recombinant inhibitors can be carried out according to methods familiar to a person skilled in the art.
For this a nucleic acid (preferably DNA) is firstl
REFERENCES:
patent: 4902623 (1990-02-01), Dowdle
Joubert et al. "The primary structure of the inhibitor of tissue plasminogen activator . . . " Thrombosis and Haemostasis 57(3), 356-360, Jun. 3, 1987.
Kouzuma, Y., "Isolation and primary structure of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds", Chemical Abstracts, vol. 119, No. 11, Abstract No. 111939, p. 397 (Sep. 13, 1993).
Kouzuma, Y., "Isolation and primary structure of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds", Biosci. Biotechnol. Biochem., vol. 56, No. 11, pp. 1819-1824 (1992).
Porath, J., and Olin, B., "Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials", Biochemistry, vol. 22, pp. 1621-1630 (1983).
Teixeira, A., "Synthesis and expression of a gene coding for Erythrina trypsin inhibitor (ETI)", Biochimica Biophysica Acta, vol. 1217, No. 1, pp. 16-22 (1994).
Fischer Stephan
Kohnert Ulrich
Stern Anne
Boehringer Mannheim GmbH
Nashed Nashaat T.
Wax Robert A.
LandOfFree
Use of recombinant inhibitor from Erythrina caffra for purifying does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of recombinant inhibitor from Erythrina caffra for purifying, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of recombinant inhibitor from Erythrina caffra for purifying will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-702187