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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。
Central composite design (CCD) was used to explore the optimal conditions for the prokaryotic expression of p ET-32a (+) - Fa MYB5 fusion protein. The induction temperature, induction time, the initial concentration of bacterial liquid and the final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) were taken as the four factors for investigation, and the soluble protein content was taken as the index for investigation. Expression of p ET-32a (+) - Fa MYB5 fusion protein was detected by glass bead method. Proteins were extracted and identified by SDS-PAGE gel electrophoresis. After optimization of the response surface, the engineered bacteria were cultured at 37 ° C until OD600 = 0.74 and IPTG was added to a final concentration of 0.40 mmol / L. After incubation at 25.51 ° C Under induction culture for 8 h, the highest yield of soluble protein of interest was obtained, which was 128 μg / m L. Optimal expression conditions obtained for the later purification of the soluble protein and the role of Fa MYB5 protein and homeopathic components provide a theoretical basis.