Journal: Metabolic Engineering
Article Title: Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production
Figure Lengend Snippet: Overexpression and cellular localization of SCD1 and SREBF1 in engineered cells. Western blot analysis of CHOK1SV GS-KO™ cells engineered to overexpress either SCD1 or SREBF1 genes where controls have been generated with an unmodified pcDNA3.1V5-His/TOPO vector. CHO-Ctl-1, CHO-Ctl-2 and CHO-Ctl-3 are monoclonal lines derived from the CHO-Control POOL ; CHO-SCD1 LOW , CHO-SCD1 MID and CHO-SCD1 HIGH are monoclonal cell lines isolated from CHO-SCD1 POOL and CHO-SREBF1 LOW , CHO-SREBF1 MID1 and CHO-SREBF1 MID2 are monoclonal lines isolated from CHO-SREBF1 POOL . The pools which had been engineered were used to generate cells expressing cB72.3 (CHO-Control cB72.3 , CHO-SCD1 cB72.3 and CHO-SREBF1 cB72.3 ) or an Fc fusion protein (FcFP; CHO-Control FcFP , CHO-SCD1 FcFP and CHO-SREBF1 FcFP ). Lysate samples were generated from these cells harvested at day 6 of culture from either host cells (A) or cells stably producing the recombinant products cB72.3 or FcFP where specified (B). A primary antibody with specificity for L7α was used as a loading control. (C) Cellular localization of overexpressed SCD1 and SREBF1 proteins in CHO-Control POOL , CHO-SCD1 POOL and CHO-SREBF1 POOL cell pools as determined by immunofluorescent detection using an anti-V5 antibody conjugated with a FITC secondary antibody. An anti-calnexin antibody conjugated with a TRITC secondary antibody was used to highlight the position of the ER. Images were obtained using confocal microscopy (see Data in Brief ( Budge et al., 2019 ) Table 2 for full details of antibodies used).
Article Snippet: In this study we show that overexpression of SCD1 and SREBF1 in the Lonza Biologics’ proprietary CHOK1SV GS-KO™ host cell line; a glutamine synthetase (GS) knockout host cell line, results in enhanced secreted recombinant protein yields of a number of different model biotherapeutic proteins, although this is dependent upon the amount of overexpression of the lipid metabolism modifying (LMM) genes (SCD1 and SREBF1).
Techniques: Over Expression, Western Blot, Generated, Plasmid Preparation, Derivative Assay, Isolation, Expressing, Stable Transfection, Recombinant, Confocal Microscopy