The development of the T7 expression system with a characteristic of cost-effective induction and tight regulation.

• Main Authors: wen-bin hung, 洪文濱
• Other Authors: yun-peng chao
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/59442538333401842724

碩士 === 逢甲大學 === 化學工程學系 === 89 === ABSTRACT The recombinant Eschreichia coli strain, BL21(BAD), was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To characterize this expression system, the plasmid containing the carbamoylase gene from Agrobacterium radiobacter under T7 promoter control was used. Upon induction with L-arabionse, the induced cell produced 139-fold increase in carbamoylase activity in comparison with the uninduced cell on M9 semi-defined medium augmented with glycerol. The protein produced accounts for 30% of total cell protein content. On the other hand, post 100 generations the plasmid harboring carbamoylase gene remained constantly stable in BL21(BAD), but its stability dropped to only 20-30% in BL21(DE3), a recombinant strain bearing T7 gene 1 regulated by the lacUV5 promoter on its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the first stage, the cell grown on glucose in the batch fermentation was fed with glycerol to continuously support its growth. Subsequently, L-arabinose was added to induce protein production of cells during late growth phage at the next stage. As a result, the carbamoylase yield corresponding to 5525 units was obtained, which amounts to 343-fold increase over that achieved in a shake-flask scale. Taken together, these results illustrate the practical usefulness of T7 system based on the araBAD promoter control for heterologous protein production, which is characterized by high-level expression capacity and stringent regulation. In the second part of this study, the recombinant E. coli strain, BL21(λG2), was used .The strain was developed to carry a chromosomal copy of T7 gene 1 fused to theλPRPL promoter. With the plasmid containing the carbamoylase gene under T7 promoter control, the strain was found to produce a large quantity of protein, upon the thermal shock. It was also found that the way by shifting temperature from 30℃ to 37℃ throughout the experiment appeared to the bast method to optimize recombinant protein production. moreover the fed-batch fermentation process was developed to enhance cell density and protein production. As a result, the cell density accounting for OD550 of 95, equivalent to 30 g dry cell weight pre liter, the carbamoylase yield corresponding to 10200 units was obtained, which amounts to 1180-fold increase over that achieved in a shake-flask scale.