拟南芥生长素结合蛋白ABP1 的原核表达及蛋白纯化

目的:鉴于生长素结合蛋白(Auxin Binding Protein,ABP)能与生长素特异性结合,因而探讨研究其直接用于生长素信号转导机理和生物传感器的可能性与可行性。方法:通过RT-PCR获得拟南芥生长素结合蛋白1(Auxin bing protein 1,ABP1)的全长CDS,将其克隆到原核表达载体pGEX4T-1中,成功构建pGEX4T-1-ABP1重组表达载体。经酶切、PCR及DNA测序鉴定后,将阳性质粒转化表达受体菌BL21(DE3)。加入异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导后,取样进行SDS-PAGE分析。结果:成功表达出一个分子量约为43 kD的可溶性融合蛋白,并利用GST亲和柱纯化方式得到了ABPl。结论:通过原核表达并经GST柱纯化后获得ABP1,为生长素生物传感器的研制开辟新的途径。同时为进一步研究ABP1与生长素的信号转导机制和生长素在生物传感测定技术中的研究和应用奠定基础。

Prokaryotic Expression and Protein Purification of the Auxin BindingProtein 1 in Arabidopsis

Objective: Auxin-binding protein can bind an auxins specifically,and can be used to study the mechanism of the signal transduction of auxin and bio-sensors.Methods: An auxin-binding protein gene(Auxin bing protein 1,ABP1) was obtained by RT-PCR from arabidopsis,and was cloned into the prokaryotic expression vector pGEX4T-1(pGEX4T-1-ABP1).Identified by digestion,PCR and DNA sequencing,the recombinant plasmid was transformed into the foreign bacteria BL21(DE3).The ABP1 protein was induced by IPTG and detected by SDS-PAGE.Results: The soluble fusion protein was expressed successfully with a molecular weight of 43 kDa.The protein was purified with GST affinity column.Conclusion:The prokaryotic expression and purity of GST-ABP1 protein show a way to development new auxin biosensor and also laid a foundation for the further study of auxin signal transduction and biosensor research.