Activated C1r and C1s Purification from Human Serum Protocol

Creative Biolabs is one of the well-recognized experts who are professional in building advanced platforms and technologies for a broad range of complement-related objectives. In the classical complement system, C1r and C1s are two proteases responsible for the activation and proteolytic activity of the C1 complex. Here, we describe the activated C1r and C1s purification from human serum protocol to promote your research.

Purification of Activated C1r and C1s from Human Serum

Flow chart of activated C1r and C1s purification from human serum.

Fig.1 Flow chart of activated C1r and C1s purification from human serum. (Creative Biolabs)

Published Data

Truncated C1s fragments are secreted as a consequence of the two pEDS C1S mutations. Fig.2 Structural analysis of truncated C1s fragments and corresponding SDS-PAGE results.¹

Ehlers-Danlos syndromes (EDS) encompass disorders with both clinical and genetic diversity. To investigate the molecular roles of C1r and C1s proteases in EDS, we conducted cell transfection studies, recombinant protein purification, mass spectrometry, and N-terminal analysis. C1sWT was generated by cloning a PCR amplicon of pFastBac1/C1s into pcDNA 3.1/Zeo using NheI and EcoRI, with a FLAG-Tag added at the 3' end for purification. Site-directed mutagenesis engineered the C1s Fg40 variant, starting at Cys354. Results show that HEK293-F cells stably transfected with the C1s variant plasmids secreted only a truncated 40 kDa Fg40 fragment, at minimal levels, instead of the full-length mutated C1s.

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Reference

Bally, Isabelle, et al. "Two different missense C1S mutations, associated to periodontal Ehlers-Danlos syndrome, lead to identical molecular outcomes." Frontiers in Immunology 10 (2019): 2962. Distributed under Open Access license CC BY 4.0, without modification.

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