Screening Of Ferment And The Inducer For High-Cell-Density-Culture Of Recombinant E.coli Expressing HrpNEcc Protein

Harpin encoded by hrpN gene in Erwinia carotovora subsp. carotovora can induce hypersensitive reaction and systemic acquired resistance on non-host plants. In the prophase, the hrpN gene was cloned into the secretion-expression vector pet30a(+), then the recombinant plasmids were transformed into E.coli BL21(DE3).The studies of recombinant E.coli BL21(DE3) culture condition showed that the follow conditions were optimize: temperature as 37℃; medium pH as 7.0; the inoculation volume was 5%; medium volume was 100ml in 500ml Erlenmeyer flask.The fermentation conditions of recombinant Escherichia coli BL21(DE3)/ pET30a(+)hrpNEcc was investigated with shaking, the optimized nutrient medium was TB medium. Orthogonal experiment showed that glucose couldn't promote cell grow and protein express. Adjustment contents of TB had no effect on cell growth and HrpNEcc expression. The result displayed optimum ration of Tryptone to Yeast extract was 1:2.While screening inducer for high cell density fermentation of recombinant Escherichia coli, the TB medium without adding any exogenous inducer was used as a control, surprisingly, the results showed that HrpNEcc protein was highly expressed. Further analysis showed that the amount of HrpNEcc protein in the TB medium reached about 301.45mg/L without adding any exogenous inducer, and the protein expressed increased 72.43% than that was cultured in LB medium induced by IPTG. Further studies indicated that HrpNEcc protein was induced by some compounds in yeast extract, the amount of expressed HrpNEcc protein greatly affected by the sources of yeast extract.The results showed that the lactose could be instead of IPTG to induce HrpNEcc protein express as exogenous inducer, and both the adding time and concentration of lactose influenced on HrpNEcc protein expression. The result showed that the HrpNEcc protein yield reached 417.60mg/L by adding 3g/L exogenous inducer lactose. Double factor (lactose adding time and concentration) test revealed that HrpNEcc yield reached the highest when adding lactose at 2h (the prophase of log-phase) in the concentration of 3g/L.Bioreactor culture test in 7L NBS bioreactor fermenting results also showed that the optimal medium was TB medium. After 25h feeding, the cell optical density at 600nm(OD₆₀₀) was 43.26, the wet cell weight(WCW) reached 57.24g/L, the total cellular protein yield reached 6.546g/L, and the HrpNEcc protein was 3.29g/L, which was about 50.2% of total cellular protein.