Reactivation of C1-inhibitor polymers by denaturation and gel-filtration chromatography

Analytical Biochemistry
M Gauthier, P A Patston

Abstract

C1-inhibitor is a proteinase inhibitor in the serpin family. It is an important inhibitor of complement C1, plasma kallikrein, and factor XIIa, and as such is involved in regulating inflammatory pathways. Studies on the plasma-derived protein are hampered by the relative ease with which the protein converts to an inactive state on storage, under mild denaturing conditions, or by incubating in some unfavorable buffers. This inactivation is caused by formation of soluble polymers which can be visualized on native electrophoresis. In order to facilitate studies on both the plasma-derived protein and recombinant variants planned for the future, it was necessary to devise a method for the rapid reactivation of the polymers in high yield. It was found that nonionic detergents did not dissociate the polymers, but they were readily dissociated in 0.1% SDS. Treatment with 0.1% SDS followed by rapid removal of the SDS and refolding on an FPLC Superose 6 column allowed for recovery of about 15% of the protein in the active monomeric form. Eighty-five percent eluted as a range of higher order polymers. Using 8 M urea as the denaturant a 25% yield of active monomer was recovered. However, with 6 M guanidine hydrochloride as the denaturant, ...Continue Reading

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Citations

Oct 7, 2004·Current Opinion in Biotechnology·Alois JungbauerRobert Schlegl
Nov 21, 2002·Blood Coagulation & Fibrinolysis : an International Journal in Haemostasis and Thrombosis·E W BrownP A Patston
Dec 20, 2018·Frontiers in Immunology·Michele MuttiValéria Szijártó

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