Thiophilic adsorption: rapid purification of F(ab)2 and Fc fragments of IgG1 antibodies from murine ascitic fluid

Yurov, Gleb K.; Neugodova, Galina L.; Verkhovsky, О.A.; Naroditsky, Boris S.

doi:10.1016/0022-1759(94)90140-6

Cited by 19 publications

(1 citation statement)

References 13 publications

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“…In general, TAC offers advantages such as neutral pH, mild elution conditions and high capacity. Efficient isolation could be demonstrated for human and mouse immunoglobulins as well as their fragments like Fabfragments and single chain antibody fragments [16][17][18][19][20], 2 -macroglobulins and complement proteins C3 and C4 [21]. Employing TAC, significant enrichment from cobra venom as well as from supernatants of different types of transfected mammalian cells could be demonstrated.…”

Section: Introductionmentioning

confidence: 96%

Purification of Native and Recombinant Cobra Venom Factor Using Thiophilic Adsorption Chromatography

Kölln

Braren²,

Bredehorst³

et al. 2007

PPL

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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.

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