High Cell Density Culture Of The Recombinant Human MT-Ⅲ In Escherichia Coli

In this thesis, the high cell-density fermentation technology of recombinant human metallothionein-â…¢(rhMT-â…¢) in Escherichia coli was researched for the first time. Conditions such as medium components, dissolved oxygen concentration, fed-batch cultivation, induction models and so on were optimized. Finally, wet cell weight of recombinant Escherichia coli in 5L fermenter was as high as 140 g/L. The expression of GST- MT-â…¢in total soluble proteins was over 23%. The recombinant protein of rhMT-â…¢was purified. The yield of rhMT-â…¢was 90 mg/L and the purity reached over 80%.The thesis includes the following four parts:1. ReviewAt first, the researches about the physicochemical properties, structure characterizations, biological functions of MT and MT-â…¢were introduced. Then the fundamental knowledge of high cell density culture of Escherichia coli was referred. At last the preparation methods of MT and the aim and value of this thesis were also briefly reviewed.2.The fermentation of recombinant Escherichia coli in shake cultureThe main components of medium were optimized with single factor experiment. The influence on the cell growth and the expression of fusion protein, including inductor concentration, expression time and the induce way and so on were researched. The result showed that it's reliable to use both IPTG inductor and lactose as inductors at the same time.3.The high cell-density fermentation of recombinant Escherichia coli in 5L fermenter Based on the optimum results of shake culture methods, the high cell-density fermentation of recombinant Escherichia coli in 5L fermenter was carried out by the DO static balance. The process conditions, such as stirring, aeration, fed-batch fermentation, inducing time were researched. When using glycerol as carbon source and adding IPTG at the middle-late log phase of cell growth, the wet cell weight could reach 115 g/L, and the expression of GST- MT-â…¢in total soluble proteins was about 23.88%. And the wet cell weight could be up to 140 g/L and the expression was 23.01% in glucose medium when using both IPTG and lactose as inductors at themiddle log phase of cell growth.4. The purification and detection of rhMT-â…¢First, the cells were disrupted by ultrasonic. The GST fusion protein was purified by glutathione-Sepharose 4B affinity column and cleaved by thrombin at the site between GST and the target sequences. The rhMT-â…¢protein was further purified and desalted by size-exclusion chromatography. The yield of rhMT-â…¢was 90 mg/L. The purity of rhMT-â…¢was measured by ELISA and reached 80%. The sulfhydry groups content of rhMT-â…¢was detected by DTNB reagents and the result was consistent with the theoretical value.