肺炎链球菌假想转录因子LytR的表达、纯化、晶体生长及优化
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摘要
本研究通过大量表达、纯化肺炎链球菌(S.pn)假想转录因子LytR,以用于晶体生长、优化及三维结构解析。以S.pn D39血清型LytR基因为DNA模板、pET-32a(+)为表达质粒、E.coli BL 21(DE 3)为表达菌株,经成功克隆、表达及纯化后,获得大量、上清表达,且纯度>90%的LytR重组蛋白(含His标签);经悬滴气相扩散法行蛋白晶体培养、初筛及优化,获得体形较大、外观规则的棒状晶体,该晶体X射线衍射能力达4.0A。本研究为假想转录因子LytR三维结构的解析及其生物学研究奠定基础。
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 0.5mmol/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 4.0A were obtained.These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.
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 0.5mmol/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 4.0A were obtained.These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.
引文
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[7]CHATFIELD C H,KOO H,QUIVEY R G Jr.The putativeautolysin regulator LytR in Streptococcus mutans plays a rolein cell division and is growth-phase regulated[J].Microbiolo-gy,2005,151(Pt 2):625-631.
[8]HAVARSTEIN L S,MARTIN B,JOHNSBORG O,et al.New insights into the pneumococcal fratricide:relationship toclumping and identification of a novel immunity factor[J].Mol Microbiol,2006,59(4):1297-1307.
[9]JOHNSBORG O,HAVARSTEIN L S.Pneumococcal LytR,aprotein from the LytR-CpsA-Psr family,is essential for nor-mal septum formation in Streptococcus pneumoniae[J].J Bac-teriol,2009,191(18):5859-5864.
[2]CLAVERYS J P,HAVARSTEIN L S.Cannibalism and frat-ricide:mechanisms and raisons d'être[J].Nat Rev Microbiol,2007,5(3):219-229.
[3]KAUSMALLY L,JOHNSBORG O,LUNDE M,et al.Cho-line-binding protein D(CbpD)in Streptococcus pneumoniae isessential for competence-induced cell lysis[J].J Bacteriol,2005,187(13):4338-4345.
[4]MASCHER T,HEINTZ M,ZAHNER D,et al.The CiaRHsystem of Streptococcus pneumoniae prevents lysis duringstress induced by treatment with cell wall inhibitors and by mu-tations in pbp2xinvolved in beta-lactam resistance[J].J Bac-teriol,2006,188(5):1959-1968.
[5]DAGKESSAMANSKAIA A,MOSCOSO M,HENARD V,etal.Interconnection of competence,stress and CiaR regulonsin Streptococcus pneumoniae:competence triggers stationaryphase autolysis of ciaR mutant cells[J].Mol Microbiol,2004,51(4):1071-1086.
[6]PETERSON S N,SUNG C K,CLINE R,et al.Identificationof competence pheromone responsive genes in Streptococcuspneumoniae by use of DNA microarrays[J].Mol Microbiol,2004,51(4):1051-1070.
[7]CHATFIELD C H,KOO H,QUIVEY R G Jr.The putativeautolysin regulator LytR in Streptococcus mutans plays a rolein cell division and is growth-phase regulated[J].Microbiolo-gy,2005,151(Pt 2):625-631.
[8]HAVARSTEIN L S,MARTIN B,JOHNSBORG O,et al.New insights into the pneumococcal fratricide:relationship toclumping and identification of a novel immunity factor[J].Mol Microbiol,2006,59(4):1297-1307.
[9]JOHNSBORG O,HAVARSTEIN L S.Pneumococcal LytR,aprotein from the LytR-CpsA-Psr family,is essential for nor-mal septum formation in Streptococcus pneumoniae[J].J Bac-teriol,2009,191(18):5859-5864.