Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease.
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 they have strong direct-acting fibrinolytic activity that could be very helpful in 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-Sepharose column 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 by circular dichroism analysis.
Selistre-de-Araujo
H S
HS
Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, Brazil. hsaraujo@power.ufscar.br
de Souza
E L
EL
Beltramini
L M
LM
Ownby
C L
CL
Souza
D H
DH
D017836:Agkistrodon / Q000378:metabolism*; D000818:Animals; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry*; D004220:Disulfides; D004229:Dithiothreitol; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006470:Hemorrhage / Q000139:chemically induced; D002479:Inclusion Bodies / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D003435:Crotalid Venoms; D004220:Disulfides; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D004229:Dithiothreitol