Expression, refolding, and in vitro activation of a recombinant snake venom pro-metalloprotease.
Metalloproteases comprise a family of Zn(2+)-endopeptidases that degrade most components of the extracellular matrix. Snake venoms are rich sources of metalloproteases, which also digest fibrinogen as well as fibrin, and in some cases, induce hemorrhage. A few low-molecular weight snake venom metalloproteases (svMPs) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity. This property could be very helpful in thrombosis therapy. ACLF is a fibrinolytic, non-hemorrhagic metalloprotease from the venom of the North American snake Agkistrodon contortrix laticinctus. We have developed an expression system for production of a recombinant pro-ACLF from a clone (ACLPREF) isolated from a venom gland cDNA library. The coding region including both the pro-enzyme domain and the mature protein domain was amplified by PCR and subcloned into the pET28a vector and the new plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at 37 degrees C led to the overexpression of an insoluble 48kDa protein after induction with 1.0mM IPTG. The expressed protein was recovered from inclusion bodies with 6M buffered urea and purified by affinity chromatography under denaturing conditions. After dithiothreitol treatment, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The pro-enzyme underwent auto-activation during refolding and it was active on fibrinogen and on a synthetic substrate. To control the activation step, the denaturing agent was rapidly removed to keep the protein in an unprocessed form, followed by later addition of Ca(2+) and Zn(2+) ions. This allowed controlling the enzyme activation, when it is needed.
Ramos
Oscar H P
OH
Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP 13565-905, Brazil.
Carmona
Adriana K
AK
Selistre-de-Araujo
Heloisa S
HS
D017836:Agkistrodon; D000818:Animals; D003435:Crotalid Venoms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D003435:Crotalid Venoms; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases