Recombinant cobra venom factor.

Cobra venom factor (CVF) is the complement-activating protein from cobra venom. CVF is a three-chain protein that functionally resembles C3b, the activated form of complement component C3. Like C3b, CVF forms a C3/C5 convertase with factor B in the presence of factor D and Mg(2+). Although CVF exhibits functional activity of C3b, it structurally resembles the C3b degradation product C3c, which is not able to form a C3/C5 convertase. CVF has become an important research tool to decomplement laboratory animals in order to study the role of complement in host defense, immune response, and pathogenesis of disease. As the Asian cobras of the Naja species are on the list of endangered species, cobra venom as the source for CVF has become increasingly difficult to obtain. Methods have been developed to recombinantly produce active forms of CVF. This manuscript reviews the production of recombinant pro-CVF using both prokaryotic and eukaryotic expression systems. The recombinant production of pro-CVF in two insect cell expression systems (baculovirus-infected Sf9 Spodoptera frugiperda cells, stably transfected S2 Drosophila melanogaster cells) generates three forms of pro-CVF: single-chain pro-CVF resembling pro-C3, a two-chain form of pro-CVF resembling C3, and another two-chain form of pro-CVF resembling C3b. All three forms of pro-CVF exhibit functional activity of mature, natural CVF. Recombinant pro-CVF supports the activation of factor B in the presence of factor D and Mg(2+), forms a bimolecular convertase pro-CVF,Bb that exhibits cleaving activity for both C3 and C5, and depletes the serum complement activity. The activity of pro-CVF and the resulting C3/C5 convertase is indistinguishable from CVF and the CVF,Bb convertase. Recombinant production of functionally active forms of pro-CVF ensures the availability of an important research reagent for future research involving complement depletion. The experimental systems to recombinantly produce active forms of CVF will also be invaluable for studies to delineate the structure/function relationship of CVF and its differences from C3, and to generate human C3 derivatives with CVF-like function ("humanized CVF") for therapeutic complement depletion.

Structure and function of recombinant cobra venom factor.

Cobra venom factor (CVF) is the complement-activating protein from cobra venom. It is a structural and functional analog of complement component C3. CVF functionally resembles C3b, the activated form of C3. Like C3b, CVF binds factor B, which is subsequently cleaved by factor D to form the bimolecular complex CVF,Bb. CVF,Bb is a C3/C5 convertase that cleaves both complement components C3 and C5. CVF is a three-chain protein that structurally resembles the C3b degradation product C3c, which is unable to form a C3/C5 convertase. Both C3 and CVF are synthesized as single-chain prepro-proteins. This study reports the recombinant expression of pro-CVF in two insect cell expression systems (baculovirus-infected Sf9 Spodoptera frugiperda cells and stably transfected S2 Drosophila melanogaster cells). In both expression systems pro-CVF is synthesized initially as a single-chain pro-CVF molecule that is subsequently proteolytically processed into a two-chain form of pro-CVF that structurally resembles C3. The C3-like form of pro-CVF can be further proteolytically processed into another two-chain form of pro-CVF that structurally resembles C3b. Unexpectedly, all three forms of pro-CVF exhibit functional activity of mature, natural CVF. Recombinant pro-CVF supports the activation of factor B in the presence of factor D and Mg2+ and depletes serum complement activity like natural CVF. The bimolecular convertase pro-CVF,Bb exhibits both C3 cleaving and C5 cleaving activity. The activity of pro-CVF and the resulting C3/C5 convertase is indistinguishable from CVF and the CVF,Bb convertase. The ability to produce active forms of pro-CVF recombinantly ensures the continued availability of an important research reagent for complement depletion because cobra venom as the source for natural CVF will be increasingly difficult to obtain as the Indian cobra is on the list of endangered species. Experimental systems to express pro-CVF recombinantly will also be invaluable for studies to delineate the structure and function relationship of CVF and its differences from C3 as well as to generate human C3 derivatives with CVF-like function for therapeutic complement depletion ("humanized CVF").