家蚕G蛋白γ1亚基(BmGγ1)的克隆及其谷胱甘肽硫转移酶融合蛋白的表达与纯化

Cloning of BmGγ1 Gene and Expression and Purification of the Coding Protein Fused With GST

异三元G蛋白是真核细胞感知外界信号后将信号传递到胞内的重要分子,参与生物体广泛的信号转导。为了研究家蚕体内G蛋白的生理功能及其作用机制,运用生物信息学方法预测了家蚕G蛋白γ1亚基(Gγ1)的序列,设计引物验证预测序列后,克隆了家蚕Gγ1的序列,再通过酶切克隆至表达载体pET-41b(+)后,导入E.coliBL21宿主菌中,经异丙基β-D-硫代半乳糖苷(IPTG)诱导表达重组谷胱甘肽硫转移酶(glutathione s-transferase,GST)融合蛋白,并亲和层析纯化表达产物。家蚕Gγ1重组GST融合蛋白经SDS-PAGE电泳和Western blot分析,在分子质量约36 kD处出现特异性蛋白条带,重组蛋白经GST亲和层析柱纯化后,得到了高纯度的融合蛋白,说明已经成功克隆到家蚕Gγ1基因,并在E.coliBL21中高效表达。

The heterotrimeric GTP-binding proteins are vital molecules in eukaryotic cell signal transduction, and they mediate extracellular signals to pass down to the downstream effectors. In order to investigate the physiological function of the silkworm G protein, a novel G protein gamma subunit gene, termed as BmGγ1, was amplified from silkworm total RNA by using methods of in silico cloning and RT-PCR. Then, the ORF of BmGγ1 subcloned into the expression vector pET-41b（＋） by enzyme digestion and ligation, resulted in recombinant expression vector, pET-41b（＋）-Gγ1. pET-41b（ ＋）-Gγ1 was transformed into E. coil BL21. Recombinant GST fusion protein was expressed by the induction of IPTG. SDS-PAGE and Western blot analysis results showed that a specific protein band of about 36 kD appeared. Through GST-affinity column, the GST-fused protein of high purity could be obtained. These results indicated that BmGγ1 was cloned successfully and expressed efficiently in E. coil BL21.