乳糖替代IPTG诱导脱色酶TpmD基因在大肠杆菌中的高效表达

Over-expression of Highly Active Triphenylmethane Dyes Decolorization Enzyme (TpmD) Induced by Lactose Instead of IPTG in Escherichia coli BL21 (DE3)

摘要:

本文考察了乳糖代替IPTG诱导三苯基甲烷类染料脱色酶TpmD在大肠杆菌BL21(DE3)中表达的可行性, 分别对用乳糖作为诱导剂时的诱导时机、乳糖浓度、诱导持续时间和添加方式进行优化并与IPTG诱导的差异等方面进行了比较分析, 确定了乳糖诱导的最佳条件。结果表明, 在工程菌对数生长中期(OD600约为0.8)添加终浓度为0.4 mmol/L的乳糖诱导6 h的条件下能获得最大量的目的蛋白和菌体量。由于乳糖可以作为碳源被菌体利用, 分批添加乳糖效果优于一次性添加。乳糖诱导条件下目的蛋白表达量占总蛋白的35.62%, 与IPTG诱导条件下的35.03%无明显差异。乳糖诱导后外源蛋白的表达时间有所滞后, 但收获的菌体量高于IPTG诱导, 显示出了乳糖同样是一种T7启动子的廉价高效诱导剂, 可以代替昂贵的IPTG用于脱色酶TpmD的规模化发酵, 同时也为其他重组蛋白的生产提供了有益的参考和借鉴。

关键词:乳糖诱导, IPTG, TpmD, T7启动子

Abstract:

The feasibility of expression of TpmD in recombinant E .coli BL21(DE3) induced by lactose instead of IPTG was investigated. The factors affecting the induction of target gene expression such as the optimal time point for induction, the concentration and addition mode of the inducer (lactose) and the induction time were determined. It is established that the optimal induction method is to add 0.4 mmol/L (final concentration) lactose at the mid-log-phase of cell growth, (OD600≈0.8) and incubate at 37°C for 6 h. It would be better to add the lactose in 4 batches (0.1 mmol/L per batch), because lactose can be used as a carbon source by E. coli BL21(DE3). The production of TpmD enzyme induced by lactose was about 35.62% of the bacterial total protein which was no significantly different from that induced by IPTG(≈35.03%), and the expression of TpmD was a little slower than that induced by IPTG. However, the final biomass induced by lactose was higher than that induced by IPTG. These results suggested that the lactose is as effective as IPTG for T7 promoter induction and should be easily scaled up for industrial production of recombinant protein with lower cost.

Key words:Lactose introduction, IPTG, TpmD, T7 promoter