| 초록 |
Here we report that an E. coli vector system, capable of improving noticeably the yield of protein expression, was newly devised. It was assumed that the accumulation of acetate has been troublesome on protein production and cell growth at the high density culture of E.coli since it has lipophilic and acidic properties. Various trials such as mutation of genes coding enzymes of acetate pathway or redirection of carbon flux from pyruvate to another metabolite have been done up to now. In this study, we used antisense gene coding an enzyme of major acetate pathway to regulate acetate formation. This strategy has been targeted on the construction of microbial system that is able to use efficiently its energy and material resources for protein expression by antisense down-regulation of overproducing acetate at the specific time. Three kinds of plasmid with gene each coding antisense ack (acetate kinase), pta (phosphotransacetylase), and both were constructed. Green fluorescent protein (GFP) was used as a foreign protein for monitoring protein expression level under trc promoter. These plasmid systems were tested for several host strains. The protein expression level increased up to five folds of the control system with only GFP under trc promoter. The cell growth level also showed better tendency in the case of plasmid with antisense. Acetate decreased to below 50% of control system that does not have antisense gene at the stationary phase. |
