Spence J, Georgopoulos C
Department of Cellular, Viral, and Molecular Biology, University of Utah Medical Center, Salt Lake City 84132.
J Biol Chem. 1989 Mar 15;264(8):4398-403.
As a preliminary to the understanding of the function of the highly conserved Escherichia coli heat shock protein HtpG, the protein was purified and partially characterized. The htpG gene was subcloned into the inducible expression vector, pT7-6. Upon induction, the HtpG protein accumulated to approximately 30% of the total protein in the cell. A purification scheme was devised which involved column chromatography on DEAE-cellulose, hydroxylapatite, and Sephacryl S-200. The amino acid composition of the purified protein corresponded closely with the predicted amino acid composition derived from the DNA sequence, and the sequence of the 8 amino-terminal residues matched the predicted sequence exactly. The molecular weight of the denatured protein is 65,500 and the native molecular weight is 144,620, as calculated by using both the Stokes radius and the sedimentation coefficient. As the molecular weight predicted from the DNA sequence is 71,429, this indicates the HtpG protein is a dimer. The HtpG protein was found to be a phosphoprotein. Thus, HtpG is structurally similar to its eukaryotic homologue, hsp83, which is also a phosphoprotein and a dimer.
作为了解高度保守的大肠杆菌热休克蛋白HtpG功能的前期工作，对该蛋白进行了纯化并进行了部分特性分析。将htpG基因亚克隆到可诱导表达载体pT7-6中。诱导后，HtpG蛋白在细胞中积累至总蛋白的约30%。设计了一种纯化方案，包括在DEAE-纤维素、羟基磷灰石和Sephacryl S-200上进行柱色谱。纯化蛋白的氨基酸组成与从DNA序列推导的预测氨基酸组成密切相符，并且8个氨基末端残基的序列与预测序列完全匹配。通过使用斯托克斯半径和沉降系数计算，变性蛋白的分子量为65,500，天然分子量为144,620。由于从DNA序列预测的分子量为71,429，这表明HtpG蛋白是二聚体。发现HtpG蛋白是一种磷蛋白。因此，HtpG在结构上与其真核同源物hsp83相似，hsp83也是一种磷蛋白且为二聚体。
