巴西固氮螺菌单链DNA结合蛋白的表达、纯化及生化特性分析

Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae.

作者信息

Vernal Javier, Serpa Viviane I, Tavares Carolina, Souza Emanuel M, Pedrosa Fábio O, Terenzi Hernán

机构信息

Laboratório de Expressão Gênica, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil.

出版信息

Protein Expr Purif. 2007 May;53(1):195-200. doi: 10.1016/j.pep.2006.11.018. Epub 2006 Dec 5.

DOI:10.1016/j.pep.2006.11.018

PMID:17223357

Abstract

An open reading frame encoding a protein similar in size and sequence to the Escherichia coli single-stranded DNA binding protein (SSB protein) was identified in the Herbaspirillum seropedicae genome. This open reading frame was cloned into the expression plasmid pET14b. The SSB protein from H. seropedicae, named Hs_SSB, was overexpressed in E. coli strain BL21(DE3) and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein. The apparent molecular mass of the native Hs_SSB was estimated by gel filtration, suggesting that the native protein is a tetramer made up of four similar subunits. The purified protein binds to single-stranded DNA (ssDNA) in a similar manner to other SSB proteins. The production of this recombinant protein in good yield opens up the possibility of obtaining its 3D-structure and will help further investigations into DNA metabolism.

摘要

在巴西固氮螺菌（Herbaspirillum seropedicae）基因组中鉴定出一个开放阅读框，其编码的蛋白质在大小和序列上与大肠杆菌单链DNA结合蛋白（SSB蛋白）相似。该开放阅读框被克隆到表达质粒pET14b中。来自巴西固氮螺菌的SSB蛋白，命名为Hs_SSB，在大肠杆菌BL21(DE3)菌株中过表达并纯化至同质。质谱数据证实了该蛋白质的身份。通过凝胶过滤估计天然Hs_SSB的表观分子量，表明天然蛋白质是由四个相似亚基组成的四聚体。纯化后的蛋白质以与其他SSB蛋白相似的方式与单链DNA（ssDNA）结合。这种高产率的重组蛋白的产生为获得其三维结构开辟了可能性，并将有助于进一步研究DNA代谢。