长双歧杆菌BL0033与BL0034的相互作用研究  

Interaction analysis between BL0033 and BL0034 in Bifidobacterium longum

作　　者：郭燕红[1,2] 刘大伟[2] 孙忠科[2] 邵长林[1,2] 何湘[2] 王雪松[2] 姜铮[2] 赵江丽[2] 刘威[2] 魏晓[2] 廖祥儒[1] 袁静[2] 

机构地区：[1]江南大学生物工程学院,江苏无锡214036 [2]军事医学科学院疾病预防控制所,北京100071

出　　处：《军事医学科学院院刊》2010年第6期551-555,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家高技术研究发展计划(863计划)(2007AA02Z118);国家自然科学基金项目(30771809)

摘　　要：目的克隆表达B.longum NCC2705果糖ABC转运系统中BL0033、BL0034及其截短突变体,验证两蛋白的体外相互作用,并确定介导彼此相互作用的功能区域。方法将bl0033与bl0034基因克隆至载体pGEX-4T-1和pET32a并在E.coli中表达,采用谷胱甘肽-Sepharose 4B和镍柱分别对GST和His融合的蛋白进行纯化,GST pull-down验证BL0033与BL0034蛋白之间的相互作用。为了进一步确定BL0033和BL0034的作用位点,根据BL0033和BL0034基序特征构建截短突变体,GST pull-down实验检测截短体与完整蛋白结合的能力,确定介导相互作用的区域。结果在大肠杆菌中实现了融合蛋白的可溶性表达,经亲和层析获得了融合蛋白。GST pull-down实验证实BL0033与BL0034存在相互作用,其中BL0033的截短体1～23 aa能与BL0034结合,而截短体36～314 aa丧失了与BL0034的结合能力;BL0034的3个截短体中BL0034/8～244 aa、BL0034/33～220 aa能与BL0033相互作用,而BL0034/289～481 aa与BL0033没有作用。结论证实了BL0033与BL0034之间的相互作用,并确定BL0033的1～23 aa基序与BL0034的33～220 aa基序是这种相互作用的结构域,为进一步研究B.longum NCC2705果糖ABC转运系统的分子机制奠定了理论基础。Objective To express and purify BL0033,BL0034 and their truncated mutants in fructose ABC transporter system of Bifidobacterium longum NCC2705,to verify the interaction between BL0033and BL0034,and to further determine the function region which mediates protein interaction.Methods Both bl0033 and bl0034 were cloned into vectors pGEX-4T-1 and pET32a and expressed by E.coli BL21.GST fusion protein and His fusion protein were purified by glutathione-Sepharose 4B beads and nickel column respectively.Interaction between BL0033 and BL0034 was analyzed by GST-pull down method.According to their function motif,truncated mutants of BL0033 and BL0034 were designed,cloned and expressed before GST pull-down assay was used to determine the interaction motif.Results The soluble fusion protein was expressed effectively in BL21 strain and purified by affinity chromatography.Obvious interaction was detected between BL0033 and BL0034.Furthermore,the functional area of BL0033 which could interact with BL0034 was the first motif 1-23 aa;truncated mutants 33-220 aa of BL0034 could strongly interact with BL0033 while the other two truncated mutants scarcely interacted with BL0033.Conclusion Strong interaction exists between BL0033 and BL0034,in which and motif 1-23 aa of BL0033 and motif 33-220 aa of BL0034 play an important role.This finding helps molecular mechanism studies on fructose ABC transporter of B.longum NCC2705.

关 键 词：GST pull-down BL0033 BL0034 截短突变体 蛋白间相互作用 

分 类 号：Q78[生物学—分子生物学]