Jepson S G, Close T J
Department of Botany and Plant Sciences, University of California, Riverside 92521-0124, USA.
Protein Expr Purif. 1995 Oct;6(5):632-6. doi: 10.1006/prep.1995.1083.
A maize dehydrin protein (Dhn1) containing 167 amino acids with a predicted molecular weight of 17.0 kDa was produced in the Escherichia coli overexpression strain BL21 (DE3)pLysS. Site-directed mutagenesis was used to construct a plasmid with a protein coding region corresponding exactly to the original cDNA. Protein production was induced by IPTG. Dhn1 was enriched from total soluble protein by heat-fractionation and centrifugation and then purified by sequential cation exchange and hydrophobic interaction chromatography. The purified protein was visualized by SDS-PAGE and immunoblot analysis using a polyclonal antibody to the dehydrin consensus region. Expression in E. coli resulted in approximately 1.2 mg of purified protein per liter of induced culture.
一种含有167个氨基酸、预测分子量为17.0 kDa的玉米脱水蛋白（Dhn1）在大肠杆菌过表达菌株BL21(DE3)pLysS中产生。采用定点诱变构建了一个蛋白质编码区与原始cDNA完全对应的质粒。通过IPTG诱导蛋白质表达。通过热分级分离和离心从总可溶性蛋白中富集Dhn1，然后通过连续的阳离子交换和疏水相互作用色谱法进行纯化。使用针对脱水蛋白共有区域的多克隆抗体，通过SDS-PAGE和免疫印迹分析对纯化的蛋白质进行可视化。在大肠杆菌中的表达导致每升诱导培养物产生约1.2 mg的纯化蛋白。
