LIU Qi-gang, DAI Yun-jian, ZHANG Yong-xia, WANG Bao-cheng, WANG Ming-rong
China Biotechnology. 2012, 32(11): 23-28.
Objective: An anti-IgE scFv was used to investigate the influence of different host strains, growth media, and culture conditions on the high-level expression of soluble single-chain antibody fragment in E. coli periplasm. Method: Three engineered bacterial strains, Rosetta (DE3), BL21(DE3), and SoluBL21(DE3), were constructed and effects of different carbon sources, nitrogen sources, growth media, and culture conditions on the amount of expression of soluble anti-IgE scFv were evaluated. Results: The expression level of pET-IgE26, a plasmid that carried anti-IgE scFv sequence, was significantly enhanced in the novel host strain SoluBL21(DE3), compared to that in traditional Rosetta (DE3) and BL21(DE3) host strains. After optimization of carbon and nitrogen sources, growth media and culture conditions, the optimal growth media for high-level expression of soluble recombinant antibodies in SoluBL21(DE3) engineered strain was found to be M9 medium with 0.5% glucose, 0.6% bacto casitone, and 0.02% trace elements. The best culture condition was defined as growing overnight culture in LB medium at 37℃, until OD600 reached approximately 3.0, then inoculate the optimal growth media with the overnight culture at 5% inoculum concentration, shake at 37℃, 260r/min for 3.5h. For induction, lower the temperature to 25℃ when OD600 is between 1.5～1.8, add 0.1mmol/L IPTG, and incubate at 220r/min for 16h. ELISA detected that the expression of soluble anti-IgE scFv increased by 5-fold after optimization. Conclusion: Expression of recombinant scFv in E coli periplasm can be significantly enhanced through optimization of host strain types, growth media, and culture conditions. The study provides technical support for scale-up production of anti-IgE scFv and insights into the production of recombinant micromolecular antibody by E. coli.
