Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography.
The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.
Kölln
Johanna
J
Institut für Biochemie und Lebensmittelchemie, Abteilung für Biochemie und Molekularbiologie, Universität Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
Braren
Ingke
I
Bredehorst
Reinhard
R
Spillner
Edzard
E
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D051056:Complement Inactivating Agents / Q000032:analysis / Q000302:isolation & purification; D006224:Cricetinae; D003412:Cricetulus; D004546:Elapid Venoms / Q000737:chemistry / Q000302:isolation & purification*; D017815:Elapidae; D004591:Electrophoresis, Polyacrylamide Gel; D011994:Recombinant Proteins / Q000302:isolation & purification
D051056:Complement Inactivating Agents; D004546:Elapid Venoms; D011994:Recombinant Proteins; C016310:cobra venom factor