变异链球菌核糖-5-磷酸异构酶A的表达、纯化与鉴定 .pdfVIP

变异链球菌核糖-5-磷酸异构酶A的表达、纯化与鉴定

武文琦;丛旭珍;殷爱红;胡家;翟方丽;李慎涛

【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(ribose5-

phosphateisomeraseA,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.%ObjectiveToexpress,purifyandcharacterizetheribose5-

phosphateisomeraseA(rpiA)fromStreptococcusmuians.MethodsA

DNAfragmentencodingS.mutansribose5-phosphateisomeraseAwas

amplifiedbyPCRusingthegenomicDNAofStreptococcusmutansUA159

asatemplate.ThePCRproductwasclonedintovectorpGEX-6p-l.The

constructcarryingthecodingDNAsequenceofrpiAfusedwithGSTwas

transformedintoE.coliBL21(DE3),andthefusionproteinwasexpressed

byinductionwithIPTG.Therecombinantproteinwaspurifiedbyaffinity

chromatographyandionexchangechromatography.Thepurifiedtarget

proteinwasidentifiedbySDSandMALDI-TOFMS.ResultsS.

mutansribose5-phosphateisomeraseAwassuccessfullyexpressedinE.

coliinsolubleform.Afteraseriesofpurifications,wegotthe

recombinantproteinwiththepurityhigherthan95%.Theproductwas

identifiedtobeS.muiansribose5-phosphateisomeraseAbySDS

andMALDI-TOF-TOFMS.Conclusion5.muiansribose5-phosphate

isomeraseAwassuccessfullyexpressedinE.coli.Aneffectivepurification

protocolwasestablished.RecombinantrpiAwithapurityhigherthan95%

wasobtained,whichprovidedabasisforthefurtherstudiesofthe

biologicalactivitiesandfun