Shuffle t7 express e coli cells

An anti‐c‐met humanized scFv23 was cloned into a pET‐22b vector in Shuffle T7 express E. coli cells (New England Biolabs, Beverly, MA). Transformants were cultured at 30° to an optical density (OD) of 0·8 at 600 nm, induced with isopropyl β‐d‐1‐thiogalactopyranoside and incubated overnight at 16°. Cell pellets were resuspended, sonicated and centrifuged at 28 700 g for 30 min. The scFv was purified from supernatants using DEAE–Sepharose anion exchange chromatography, followed by Protein A affinity chromatography, through binding the VH region, and size exclusion chromatography.22 Protein concentrations were determined by measuring the absorbance at 280 nm using an extinction coefficient of 58 580/M/cm.
For SEC‐MALLS (size exclusion chromatography coupled with multi‐angle laser light scattering) analysis the SEC column outlet was connected to a Dawn Helios MALLS photometer (Wyatt, Santa Barbara, CA) followed by an OptiLab T‐rEX differential refractometer (Wyatt). Data were processed using the wyatt‐qels software (Wyatt, Santa Barbara, CA). Samples were processed by staff at the Biomolecular Analysis Facility at the University of Manchester.

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).