Recombinant Expression and Purification of C5a

Recombinant hC5a (mutant Cys704Arg), mC5a and mC5a-desArg were expressed recombinantly in bacteria23 (link). All proteins were expressed as fusions with an N-terminal thioredoxin tag (Trx-A) followed by a hexahistidine tag and a TEV protease cleavage site (Trx-His₆-TEV-C5a), in Shuffle T7 Express E. coli cells (New England Biolabs). The proteins were purified using a two-step Ni-column affinity chromatography, including removal of the affinity-tag by overnight incubation with TEV protease, followed by a cation exchange chromatography on a Source 15S column (GE Healthcare Life Sciences). The final protein buffer was adjusted to 20 mM HEPES, pH 7.5, 150 mM NaCl before flash freezing in liquid nitrogen and storage at −80 °C until use. Mutants of hC5a and mC5a were generated using the Quick-Change Lightning Site Directed Mutagenesis Kit from Agilent Technologies and all mutants were expressed and purified using the same protocol as for native proteins. Human C3a was expressed recombinantly following the same protocol as for the C5a proteins50 (link).

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Yatime L., Maasch C., Hoehlig K., Klussmann S., Andersen G.R, & Vater A. (2015). Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer. Nature Communications, 6, 6481.

Publication 2015

Shuffle T7 Express E. coli cells Source 15S column Quick-Change Lightning Site Directed Mutagenesis Kit

Affinity chromatography Buffer Cells Chromatography Cleavage E coli Hepes Hexahistidine Human Nacl Nitrogen Proteins Site directed mutagenesis Tev protease Thioredoxin

Corresponding Organization :

Other organizations : Aarhus University, Noxxon Pharma (Germany)

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Variable analysis

independent variables

Recombinant hC5a (mutant Cys704Arg)
MC5a-desArg

dependent variables

Not explicitly mentioned

control variables

Expression of proteins as fusions with an N-terminal thioredoxin tag (Trx-A) followed by a hexahistidine tag and a TEV protease cleavage site (Trx-His6-TEV-C5a) in Shuffle T7 Express E. coli cells
Purification using a two-step Ni-column affinity chromatography, including removal of the affinity-tag by overnight incubation with TEV protease, followed by a cation exchange chromatography on a Source 15S column
Final protein buffer adjusted to 20 mM HEPES, pH 7.5, 150 mM NaCl before flash freezing and storage at −80 °C
Expression and purification protocol for mutants of hC5a and mC5a was the same as for native proteins

controls

Positive control: Human C3a expressed recombinantly following the same protocol as for the C5a proteins

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