嗜热栖热菌OT3中一种超嗜热古菌组蛋白的重组表达与特性分析

Recombinant expression and characterization of an extremely hyperthermophilic archaeal histone from Pyrococcus horikoshii OT3.

作者信息

Weng Liang, Feng Yan, Ji Xin, Cao Shugui, Kosugi Yoshitsugu, Matsui Ikuo

机构信息

Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China.

出版信息

Protein Expr Purif. 2004 Jan;33(1):145-52. doi: 10.1016/j.pep.2003.09.004.

DOI:10.1016/j.pep.2003.09.004

PMID:14680971

Abstract

A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli. The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868Da. Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones. The HPhA was purified to homogeneity by heat precipitation and affinity chromatography. Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA. The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65 degrees C. Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90 degrees C. The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography.

摘要

克隆、测序了嗜热古菌火球菌OT3菌株的一个类组蛋白基因PHS051，并在大肠杆菌中进行了表达。重组组蛋白HPhA编码一个由70个氨基酸组成、分子量为7868Da的蛋白质。对HPhA的氨基酸序列分析表明，它与其他古菌组蛋白和真核核心组蛋白具有高度同源性。通过热沉淀和亲和层析将HPhA纯化至同质。凝胶电泳迁移率变动分析表明，纯化的HPhA对DNA具有高亲和力。HPhA与DNA的复合物可使DNA在65℃下免受限制性内切酶TaqI的切割。圆二色光谱显示，当温度从25℃升高到90℃时，重组组蛋白HPhA的构象变得更松散。HPhA具有显著的热稳定性，尤其是在1M KCl存在的情况下，并且在极端温度下仍保留DNA结合活性，这与我们之前通过X射线晶体学分析其结构稳定性的报告一致。