Expression, purification and crystallization of RecJ from Pyrococcus furiosus

YANG Min; LI Minjun; ZHOU Huan; CHEN Yaxing; SUN Lihua; YU Feng; XU Chunyan; TANG Lin; HE Jianhua

doi:10.11889/j.0253-3219.2013.hjs.36.080103

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Expression, purification and crystallization of RecJ from Pyrococcus furiosus

更新时间：2021-03-05

- Expression, purification and crystallization of RecJ from Pyrococcus furiosus
- NUCLEAR TECHNIQUES Vol. 36, Issue 8, Article number: 080103(2013)
- 作者机构：
  
  中国科学院上海应用物理研究所
- 作者简介：
- 基金信息：
- DOI：10.11889/j.0253-3219.2013.hjs.36.080103
  CLC： Q78
- Published：01 August 2013
- 稿件说明：
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[1]杨敏,李敏军,周欢,陈亚星,孙丽华,郁峰,徐春艳,唐琳,何建华.火球菌RecJ蛋白的表达纯化及结晶[J].核技术,2013,36(08):17-21.

YANG Min, LI Minjun, ZHOU Huan, et al. Expression, purification and crystallization of RecJ from Pyrococcus furiosus[J]. Nuclear techniques, 2013, 36(8): 080103
[1]杨敏,李敏军,周欢,陈亚星,孙丽华,郁峰,徐春艳,唐琳,何建华.火球菌RecJ蛋白的表达纯化及结晶[J].核技术,2013,36(08):17-21. DOI： 10.11889/j.0253-3219.2013.hjs.36.080103.

YANG Min, LI Minjun, ZHOU Huan, et al. Expression, purification and crystallization of RecJ from Pyrococcus furiosus[J]. Nuclear techniques, 2013, 36(8): 080103 DOI： 10.11889/j.0253-3219.2013.hjs.36.080103.

摘要

RecJ蛋白是一类在细菌和古菌中高度保守的蛋白

在细胞内参与同源重组、甲基指导的碱基错配修复和碱基切除修复等过程。RecJ蛋白除了可以从单链DNA的5端降解DNA外

还可以从单链RNA的3端降解RNA。为能够从结构上阐明古菌火球菌（Pyrococcus furiosus）RecJ蛋白在细胞内的功能和其对不同底物（RNA/DNA）的降解活性的影响

本实验采用蛋白质晶体学的方法

通过蛋白质的表达纯化

晶体筛选得到了火球菌RecJ蛋白的晶体

并通过结晶条件的初步优化得到了有衍射的晶体。

Abstract

Background: RecJ

a highly conserved protein in archaea and bacterium

is a single-stranded DNA（ssDNA）-specific 5 to 3 exonuclease that plays an important role in homologous recombination

base excision repair（BER） and methyl directed mismatch repair（MMR）.And RecJ could degrade single-stranded RNA（ssRNA） from 3 to 5 specially.Purpose: RecJ from pyrococcus furiosus was expressed

purified and crystallized.The aim is to get the structure of the protein

clarify the functions of RecJ in cells and to investigate its effect on the degradation of different nucleotide substrates（DNA/RNA）.Methods: In order to get the crystal of RecJ

E.coli was chosed to express the protein.And the protein was purified by metal-chelating chromatography and anion exchange chromatography.The crystal was obtained by screening and the crystal with better diffraction was got by optimization.The data was collected from beamline BL17U1 at SSRF.Results and conclusions: The crystal of P.fu RecJ was got by screening

but the structure has not been achieved because of the low resolution of crystal.

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