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Titolo:
Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease
Autore:
Selistre-de-Araujo, HS; de Souza, EL; Beltramini, LM; Ownby, CL; Souza, DHF;
Indirizzi:
Univ Sao Paulo, Inst Fis Sao Carlos, Dept Fis & Informat, Sao Carlos, SP, Brazil Univ Sao Paulo Sao Carlos SP Brazil s & Informat, Sao Carlos, SP, Brazil Univ Fed Sao Carlos, Dept Ciencias Fisiol, BR-13565905 Sao Carlos, SP, Brazil Univ Fed Sao Carlos Sao Carlos SP Brazil BR-13565905 BCCarlos, SP, Brazil Oklahoma State Univ, Coll Vet Med, Dept Anat Pathol & Pharmacol, Stillwater, OK 74078 USA Oklahoma State Univ Stillwater OK USA 74078 col, Stillwater, OK 74078 USA
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 1, volume: 19, anno: 2000,
pagine: 41 - 47
SICI:
1046-5928(200006)19:1<41:ERAAOA>2.0.ZU;2-Z
Fonte:
ISI
Lingua:
ENG
Soggetto:
AGKISTRODON-CONTORTRIX-LATICINCTUS; BROAD-BANDED COPPERHEAD; AMINO-ACID-SEQUENCE; FIBRINOLYTIC ENZYME; BIOCHEMICAL-CHARACTERIZATION; HEMORRHAGIC METALLOPROTEINASES; PROTHROMBIN ACTIVATOR; MOLECULAR-CLONING; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI;
Keywords:
snake venom; metalloprotease; fibrinolysis; expression; refolding;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
35
Recensione:
Indirizzi per estratti:
Indirizzo: Selistre-de-Araujo, HS Univ Sao Paulo, Inst Fis Sao Carlos, Dept Fis & Informat, Sao Carlos, SP, Brazil Univ Sao Paulo Sao Carlos SP Brazil s, SP, Brazil
Citazione:
H.S. Selistre-de-Araujo et al., "Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease", PROT EX PUR, 19(1), 2000, pp. 41-47

Abstract

Snake venoms are rich sources of proteases that strongly affect the vascular system, by promoting blood coagulation, hemorrhage, and fibrinolysis, Hemorrhagic activity is mostly due to the enzymatic action of metalloproteases on capillary basement membrane components, such as collagen. IV, laminin,and fibronectin, A few low-molecular-weight snake venom metalloproteases (svMP) have been described as being devoid of hemorrhagic activity, but theyhave strong direct-acting fibrinolytic activity that could be very helpfulin thrombosis therapy. We have developed an expression system for production of a recombinant svMP from a cDNA (ACLPREF) coding for a small metalloprotease (ACLF) with three disulfide bonds from an Agkistrodon contortrix laticinctus (broad-banded copperhead) venom gland cDNA library. The mature protein-coding region was amplified by PCR and subcloned into the pET28a vector, and the resulting plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at either 37 or 20 degrees C led to the overexpression. of an insoluble and inactive 30-kDa protein after 1.0 mM IPTG induction, The expressed protein (rACLF) was recovered from inclusion bodies with 6 M buffered urea solution and purified on a nickel-Sepharosecolumn followed by gel filtration. chromatography, both under denaturing conditions. After treatment with dithiothreitol, protein refolding was performed by gradual removal of the denaturing agent by dialysis, The refolded recombinant protein was active in fibrin-agarose plates. The purified protein achieved a conformation similar to that of the native enzyme as judged bycircular dichroism analysis. (C) 2000 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 13/07/20 alle ore 10:09:43