变异链球菌核糖-5-磷酸异构酶A的表达、纯化与鉴定  

Expression,purification and characterization of ribose 5-phosphate isomerase A from Streptococcus mutans

作　　者：武文琦[1,2] 丛旭珍[1] 殷爱红[2] 胡家[2] 翟方丽[3] 李慎涛[1] 

机构地区：[1]首都医科大学基础医学院生物化学与分子生物学和免疫学系,北京100069 [2]首都医科大学医学实验与测试中心,北京100069 [3]青岛阜外心血管病医院检验科,青岛266034

出　　处：《首都医科大学学报》2012年第2期227-232,共6页Journal of Capital Medical University

摘　　要：目的在大肠杆菌中高效表达变异链球菌核糖-5-磷酸异构酶A(ribose 5-phosphate isomerase A,rpiA),并对表达产物进行纯化和鉴定。方法根据GenBank中变异链球菌UA159株基因组rpiA的DNA编码序列,设计PCR引物,扩增变异链球菌核糖-5-磷酸异构酶A的DNA编码序列,将其克隆至pGEX-6p-1载体中,构建重组质粒,将测序正确的重组质粒转化入大肠杆菌BL21(DE3)中,用异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达;对培养温度、IPTG用量、诱导时间等条件进行了优化;用亲和层析、离子交换层析纯化目标蛋白;用SDS-PAGE和质谱对目标蛋白进行鉴定。结果变异链球菌核糖-5-磷酸异构酶A在大肠杆菌中高效、可溶性表达,经质谱鉴定及SDS-PAGE分析,表达产物为变异链球菌核糖-5-磷酸异构酶A蛋白。经过纯化,得到纯度高达95%的变异链球菌核糖-5-磷酸异构酶A。结论成功地在大肠杆菌中高效表达了变异链球菌核糖-5-磷酸异构酶A蛋白,并建立了纯化工艺,得到高纯度的重组蛋白,为进一步研究变异链球菌属核糖-5-磷酸异构酶蛋白的生物学活性及功能奠定了基础。Objective To express,purify and characterize the ribose 5-phosphate isomerase A（rpiA） from Streptococcus mutans.Methods A DNA fragment encoding S.mutans ribose 5-phosphate isomerase A was amplified by PCR using the genomic DNA of Streptococcus mutans UA159 as a template.The PCR product was cloned into vector pGEX-6p-1.The construct carrying the coding DNA sequence of rpiA fused with GST was transformed into E.coli BL21（DE3）,and the fusion protein was expressed by induction with IPTG.The recombinant protein was purified by affinity chromatography and ion exchange chromatography.The purified target protein was identified by SDS-PAGE and MALDI-TOF MS.Results S.mutans ribose 5-phosphate isomerase A was successfully expressed in E.coli in soluble form.After a series of purifications,we got the recombinant protein with the purity higher than 95%.The product was identified to be S.mutans ribose 5-phosphate isomerase A by SDS-PAGE and MALDI-TOF-TOF MS.Conclusion S.mutans ribose 5-phosphate isomerase A was successfully expressed in E.coli.An effective purification protocol was established.Recombinant rpiA with a purity higher than 95% was obtained,which provided a basis for the further studies of the biological activities and functions of ribose 5-phosphate isomerase A.

关 键 词：变异链球菌 核糖-5-磷酸异构酶A 融合蛋白 原核表达 蛋白纯化 

分 类 号：R446.1[医药卫生—诊断学]