Jararhagin ECD-containing disintegrin domain: expression in escherichia coli and inhibition of the platelet-collagen interaction.
Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound A Disintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet-collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet-collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin-disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC(50) of 8.5 microg/ml).
Moura-da-Silva
A M
AM
Laboratório de Imunopatologia, Laboratório de Biotecnologia Molecular, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, 05503-900, Brazil. anamoura@usp.br
Línica
A
A
Della-Casa
M S
MS
Kamiguti
A S
AS
Ho
P L
PL
Crampton
J M
JM
Theakston
R D
RD
D001792:Blood Platelets / Q000187:drug effects; D003001:Cloning, Molecular; D003094:Collagen / Q000494:pharmacology; D003435:Crotalid Venoms / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D019483:Disintegrins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D000097146:Bothrops jararaca Venom
D003094:Collagen; D003435:Crotalid Venoms; D019483:Disintegrins; D008666:Metalloendopeptidases; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D000097146:Bothrops jararaca Venom