Heterologous expression and function evaluation of Gloeobacter violaceus rhodopsin in Escherichia coli / 生物工程学报

Proton-pumping rhodopsin (PPR) is a simple photosystem widely distributed in nature. By binding to retinal, PPR can transfer protons from the cytoplasmic to the extracellular side of the membrane under illumination, creating a proton motive force (PMF) to synthesize ATP. The conversion of light into chemical energy by introducing rhodopsin into nonphotosynthetic engineered strains could contribute to promoting growth, increasing production and improving cell tolerance of microbial hosts. Gloeorhodopsin (GR) is a PPR from Gloeobacter violaceus PCC 7421. We expressed GR heterologously in Escherichia coli and verified its functional activity. GR could properly function as a light-driven proton pump and its absorption maximum was at 539 nm. We observed that GR was mainly located on the cell membrane and no inclusion body could be found. After increasing expression level by ribosome binding site optimization, intracellular ATP increased, suggesting that GR could supply additional energy to heterologous hosts under given conditions.

OPTIMIZING EXPRESSION CONDITIONS OF ENGINEERED ESCHERICHIA COLI BEARING HUMANIZED ANTI-HBsAG Fab ON LABORATORY SCALE / 解放军医学杂志

Objective To investigate the best fermentation and induction models of the engineered Escherichia coli, in order to obtain the highest expression level of humanized anti HBsAg Fab. Methods The optimal condition governing the growth of the Escherichia coli, with the flask shaking method and the best fermentation and induction conditions to yield the highest production level were explored, and then E coli were grown in fermentor using the fed batch method following the same principle under flask shaking condition to ensure the best production. Results The data obtained from flask shaking conditions showed that when the induction procedure started the amount of anti HBsAg Fab would reach the highest level at mid log growth phase under the induction condition of 25℃ and 0 2% arabinose. Using the DO stat fed batch method, the OD 600 value of the culture would reach 55 2, which corresponded to 110g/L bacterial wet weight. The biological activity of Fab was proved to have well preserved. Conclusion We established the optimal production technic of HBsAg Fab, and lay a foundation to produce HBsAg Fab on a large scale