Studies on expression, purification, crystal growth and optimization of putative transcription factor LytR from Streptococcus pneumoniae.
- Author:
Xun MIN
1
;
Wen ZHONG
;
Shasha ZHAO
;
Jie DONG
;
Shanshan DONG
;
Aie ZHOU
;
Wenjuan YAN
;
Deqiang WANG
Author Information
1. Department of Medicine Laboratory, Affliated Hospital of Zunyi Medical College, Zunyi 563003, China.
- Publication Type:Journal Article
- MeSH:
Bacterial Proteins;
biosynthesis;
genetics;
isolation & purification;
Escherichia coli;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification;
Streptococcus pneumoniae;
genetics;
metabolism;
Transcription Factors;
biosynthesis;
genetics;
isolation & purification
- From:
Journal of Biomedical Engineering
2013;30(4):812-821
- CountryChina
- Language:Chinese
-
Abstract:
The aim of the present study was to obtain the crystal of transcription factor LytR of streptococcus pneumoniae for X-ray crystal structure and function analysis. The LytR gene of D39 strains from Streptococcus pneumoniae (S. pn) was cloned into the prokaryotic expression vector pET32a(+), then overexpression was obtained in the E. coli BL21 (DE3) through transformation of the recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with His-tag highly expressed by the induction of 0.5 mmol/L IPTG and was purified by a three step procedure, the purity of the purified LytR recombinant protein was over 90%. Preliminary screening of crystallization conditions was performed using the hanging-drop vapour-diffusing method with Hampton Crystal screen and PEG screen kits. The protein crystals X-ray diffraction data were collected from a single crystal and more stick crystals whose X-ray diffraction reached 4.0 A were obtained. These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.
