草莓FaMYB5基因原核表达的响应面优化

发布时间：2018-03-29 18:05

  本文选题：草莓　切入点：FaMYB　出处：《基因组学与应用生物学》2017年01期

【摘要】：利用中心组合设计(CCD)探究p ET-32a(+)-Fa MYB5融合蛋白原核表达的最优条件。以不同的诱导温度、诱导时间、菌液初始浓度和异丙基-β-D-硫代半乳糖苷(IPTG)终浓度为4个考察因素,以可溶性蛋白含量为考察指标,在大肠杆菌中表达p ET-32a(+)-Fa MYB5融合蛋白,采用玻璃珠法破菌,提取蛋白质后通过SDS-PAGE凝胶电泳鉴定表达产物。经响应面优化后,在37℃培养工程菌至菌液OD600=0.74后,加入异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度为0.40 mmol/L,在25.51℃条件下诱导培养8 h,可获得最高产量的可溶性目的蛋白,为128μg/m L。最优表达条件的获得,为后期纯化该可溶性蛋白和鉴定Fa MYB5蛋白与顺势作用元件的作用情况提供了一定的理论依据。
[Abstract]:The optimal conditions for prokaryotic expression of pET-32a (-Fa MYB5) fusion protein were investigated by central combination design. Four factors were investigated under different induction temperature, induction time, initial concentration of bacterial solution and final concentration of isopropyl- 尾 -Dthiogalactoside (IPTG). The fusion protein of pET-32a (-Fa) MYB5 was expressed in Escherichia coli with soluble protein content as the index. The protein was extracted by glass bead method, and the expressed product was identified by SDS-PAGE gel electrophoresis. The expression product was optimized by response surface optimization. After the engineering bacteria were cultured to OD600=0.74 solution at 37 鈩，

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