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Biochemical Characterization of C1r, C1s, and the C1s-C1r-C1r-C1s Tetramer
SDS-PAGE analysis is used at different purification stages to identify the C1r- and/or C1s-containing fractions and to quantify the C1r and C1s protein contents of insect cell culture supernatants before reconstitution of the recombinant tetramer. Performing this analysis under reducing conditions allows discrimination between the single-chain proenzymes and the two-chain activated proteases.
Fig.1 Flow chart of C1r, C1s, and the C1s-C1r-C1r-C1s tetramer biochemical analysis protocol.
Published Data
Fig.2 SDS-PAGE analysis illustrating C1r-mediated cleavage of zymogen C1s.1
C1r, a serine protease, triggers the activation of the complement's classical pathway, a vital component of the innate immune response against pathogens and altered-self cells. It undergoes autoactivation and subsequently cleaves and activates C1s. The cleavage of zymogen C1s by C1r was examined by incubating recombinant wild-type C1s with recombinant wild-type C1r, followed by SDS-PAGE analysis. Immunoblotting was performed using antibodies targeting a serine protease domain peptide of C1s and a polyclonal antibody for C1r. The experimental panels illustrate the time course of this process at 37°C.
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Reference
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Wijeyewickrema, Lakshmi C., et al. "Molecular determinants of the substrate specificity of the complement-initiating protease, C1r." Journal of Biological Chemistry 288.22 (2013): 15571-15580. Distributed under Open Access license CC BY 4.0, without modification.
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