Study On Recombinant E.coli DH5α Producing RNGR-Tum-5

The desertive consists of five chapters: The productivity of rNGR-Tum-5 from recombinant E.coli DH5αwas systematically studied, which includes optimization of fermentation culture medium, culture conditions in both shake flask and 5L fermenter, respectively. After high-density culture, the final cell growth of engineering bacteria OD₆₀₀ reached 16.0, the expression of rNGR-Tum-5 reached 30%, and the productivity of rNGR-Tum-5 from recombinant E.coli DH5αreached 4.80.1. Optimization of the culture media of recombinant E.coli DH5αTo improve the yield of rNGR-Tum-5 produced from recombinant E.coli, LB culture medium was chosen as the basic medium from several types of common medium. The influences of different types of carbon, nitrogen sources and trace elements in the culture medium on the yield of rNGR-Tum-5 had been investigated with shake flask method. It was found that the yield of rNGR-Tum-5 was the highest and it raised five times over that in the initial culture medium under the condition of 1% glycerol, 3% yeast extract, 1% peptone, 0.5% sodium chloride and 0.15% trace elements solution in the culture medium (w/w).2. Culture of recombinant E.coli DH5a in shake flaskThe influences of fermentation conditions such as temperature, dissolved oxygen, inoculum age, induction time, inducer concentration, expression time on the growth of bacteria and protein expression has been studied. The best culture conditions were concluded as the following: culture temperature 37℃, shaking speed 200 rpm, inoculum age OD₆₀₀ 1.0, induction at mid-logarithmic growth (OD₆₀₀= 2.0, 4 h ), IPTG concentration chosen at 0.5mmol/ L, expression time for 4h. 3. 5L fermentor culture of recombinant E.coli DH5αInitially the appropriate seed amount of 8% was confirmed. The engineering bacterial growth and protein expression in fermentor were obtained. The ability of engineering bacterial producing rNGR-Tum-5 has been raised by controlling pH, which adoptted two-stage culture method. The pH in the early stage of cell growth was different from that in the late stage of protein expression, which raised significantly the yield of rNGR-Tum-5 . The product of fermentation has been separated and determined. The target protein appeared about 10⁴ Da in the molecular weight.