CD81 Expression in Pichia pastoris

Pichia pastoris wild type strain X-33 (Invitrogen) was electroporated to integrate the hCD81 expression plasmid comprising pPICZB encoding C-terminus His₆ tagged CD81 [20 (link)]. SMA 2000 co-polymer (2:1 Cray Valley) and SZ25010 co-polymer (3:1 Polyscope) were obtained as styrene maleic anhydride and hydrolysed to styrene maleic acid forms as described previously [21 (link)]. Detergent n-dodecyl-B-D-maltoside (DDM), biotin, zeocin, yeast extract, peptone, yeast nitrogen base and agar antibiotic were purchased from ThermoFisher Scientific. Ni²⁺-NTA resin for IMAC purification was purchased from Qiagen and Bio-Rad Econo-column empty chromatography column was used. Mini EDTA-free Protease Inhibitor Cocktail Tablets from Roche Applied Science. Anti-mouse HRP-conjugated secondary antibody was purchased from Cell Signalling Technology. Ammonium formate was purchased from Sigma Aldrich. Methanol, chloroform and ultra pure water, all LC-MS grade, were purchased from Fisher, UK. Anti-CD81 antibodies 2s131, 1s337 and 1s135 were generated as described previously [22 (link)] and hybridoma supernatants were used for all ELISA experiments.