Heterologous Expression And Optimization Of HBsAg In Saccharomyces Cerevisiae

Hepatitis B surface antigen (HBsAg) is a major component of the hepatitis B vaccine,while recombinant HBsAg produced by Saccharomyces cerevisiae is a main source forgenetic engineering hepatitis b vaccine. Using genetic engineering technology, the optimizedHBsAg gene was heterologously expressed in S. cerevisiae and the expression level gotsignificantly improved by optimization at transcriptional level, translational level andpost-translational level. Then fermentation optimization was performed with flaskexperiments to further enhance the production of HBsAg by S. cerevisiae. The main contentsof this study were as follows:HBsAg was successfully expressed in S. cerevisiae with TEF1promoter. After codonoptimization to preferentially match codon frequencies of S. cerevisiae, HBsAg gene wascloned to expression vector and recombinant strain was constructed. HBsAg activity was9.82IU g-1DCW after fermentation, which suggested that HBsAg was heterologously expressedwith immune activity in S. cerevisiae using TEF1promoter. In order to simplify thepurification of expression product,6×His purification tag was added at the N terminal and Cterminal of HBsAg gene, respectively. The result of HBsAg activity showed that6×His tag atC terminal rather than N terminal had less adverse effect on HBsAg activity (8.87IU g-1DCW).HBsAg expression level was increased20times, from8.87IU g-1DCW to186.11IU g-1DCW, though optimization at transcriptional level, translational level and post-translationallevel.(1) The constitutive promoters (TEF1, TDH3, HXT7and ADH1) and induciblepromoter GAL1were used to express HBsAg, respectively, and GAL1was most favorable forHBsAg expression (46.45IU g-1DCW, increased3.87times compared to TEF1).(2)Moreover, three kozak sequences GCCACC, TACACA and AAAAAA were added,respectively, at-1to-6site of HBsAg gene to facilitate translation. The result showed thatsequence TACACA was best and HBsAg expression level was increased by37%, with anactivity of63.67IU g-1DCW.(3) When attemptting to achieve secretion expression ofHBsAg in S. cerevisiae, the most widely-used signal peptides α-factor from S. cerevisiae,reported highly efficient signal peptides ybr187w from S. cerevisiae and signal peptide nsBfrom Candida Antarctica Lipase B was used for HBsAg expression. However, no HBsAgactivity was detected in the culture supernatant, suggesting that secretory expression ofHBsAg failed with the three peptides. But HBsAg expression level was increased by31.24%with nsB signal peptide (83.56IU g-1DCW).(4) HBsAg activity was further improved1.23times by co-expression with protein disulfide isomerase (PDI) and achieved186.11IU g-1DCW.Fermentation optimization of recombinant strain was performed with flask experimentsand HBsAg expression level was improved by75.2%. In order to further increase HBsAgproduction, optimization of culture conditions, medium component and additives wereconducted with flask experiments. Optimized culture conditions were as follows:30℃, initialpH5.0, medium volume20mL (250mL), initial OD6000.5and fermentation period for72h; Optimized YPDG medium (g L-1): galactose15, glucose25, tryptone20, yeast extract10,glycerol4, sucrose6. HBsAg production was improved by75.2%by fermentationoptimization, from185.93IU g-1DCW to325.75IU g-1DCW.