华南象草PpCAD基因的原核表达研究
首发时间:2015-05-06
摘要:肉桂醇脱氢酶(Cinnamyl Alcohol Dehydrogenase, CAD)是控制木质素生物合成途径中的最后一步关键酶,对植物的木质素合成调控有十分重要的意义。本文根据已克隆的华南象草PpCAD基因cDNA序列,构建原核表达载体pGEX-PpCAD,转化BL21后诱导融合蛋白GEX-PpCAD表达,并进行诱导体系筛选,以期为PpCAD的体外酶活研究奠定基础。结果表明:在浓度为0.02 mmol/L的IPTG下,28℃诱导1h就能产生少量的可溶性融合蛋白,大小为69 kDa,除去GST大小,PpCAD约40 kDa,与预期结果相符。随着诱导时间的延长,形成包涵体。当IPTG浓度为0.1 mmol/L时,以相同条件诱导5 min就能产生包涵体融合蛋白。低温和乙醇处理可以减缓蛋白表达速率,但不能减少包涵体蛋白的产生。细菌的冻融处理可以增加包涵体蛋白的提取纯度与丰度。
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Prokaryotic Expression Analysis of A PpCAD Gene from Elephant Grass (Pennisetum purpureum cv. Huanan)
Abstract:Cinnamyl Alcohol Dehydrogenase (CAD), a multifunctional enzyme that catalyzes the last step in lignin monolignol biosynthesis, has significant meanings to plant lignification improvement. In the present study, a prokaryotic expression vector of pGEX-PpCAD was constructed based on the cDNA sequence of PpCAD gene, aiming to investigate the in vitro expression property of PpCAD protein. The E. coli strain BL21 was used as expression host. The results showed that soluble fusion protein GST-PpCAD could be induced under the condition of 0.02 mmol/L IPTG, 28℃ and 60 min, but the expression level was low. The induced fusion protein was about 69 kDa and the PpCAD was about 40 kDa, which was consistent with the prediction. Meanwhile, fusion proteinwould convert to inclusion body with increase of induction time, and it was mostly existed in inclusion body no matter how to optimaze induction conditions. Ethyl alcohol and low temperature treatment would slow down the induction of fusion protein, but had no effect on the inclusion body. Frozen treatment was conductive to the extraction of inclusion bodies.
Keywords: Pennisetum purpureum PpCAD Prokayyotic expression Inclusion body
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No.4640869920261143****
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