Pyrococcus sp.DNA聚合酶内蛋白子突变体的剪切性质  

Splicing Properties of some Mutants of Intein from Pyrococcus sp.DNA Polymerase

作　　者：石亚伟[1] 范俊虎[1] 袁静明[1] 徐明群[2] 

机构地区：[1]山西大学生物工程中心,山西太原030006 [2]New England Biolabs,beverlymausa01915

出　　处：《山西大学学报（自然科学版）》2000年第2期149-152,共4页Journal of Shanxi University(Natural Science Edition)

基　　金：国家自然科学基金研究课题!(395 70 15 4)

摘　　要：将含麦芽糖结合蛋白 -嗜热菌 DNA聚合酶内蛋白子基因的 p MI84、p MI94、p MI95三个重组突变体质粒转入 E.coli2 42 6 ,经发酵及 IPTG低温诱导表达 ,直链淀粉糖亲和纯化获得 MI84(ser1 /Ser5 38Stop)、MI94(Ser1 →Cys H1 /Ser5 38Stop)、MI95 (Ser1 → Ala1 /Ser5 38Stop)三种蛋白质前体。研究了 p H、温度、巯基试剂对内蛋白子 N末端的剪切影响。除 MI95外 ,MI84、MI94前体蛋白均能发生剪切反应 ,DTT能明显促进 MI84、MI94剪切 ,而且 MI94剪切效果优于 MI84。不同还原剂对 MI94诱导差异较小 ,温度升高能明显加快 MI94剪切反应 ,随着 p H升高也能促进 MI94剪切反应。The recombinantplasmids p MI84,p MI94and p MI95which contain maltose binding protein- DNA polymerase intein ( MI) genes are expressed in E.coil 2 4 2 6separately.The expression products are the pre- cursors MI84( Ser1/ Ser53 8Stop) ,MI94(→ Cys H1/ Ser53 8Stop) and MI95( Ser1→ Ala1/ Ser53 8Stop) respectively. The effect of temperature,p H and reducing reagents on intein spliced were studied,the results showed that MI94and MI84precursors could be cleavaged to M and Iatthe differentincubation time in37℃ ,but MI95 could not be at all.Moreover,the reducing reagents such as DTT may stimulate MI84and MI94cleavage. There is no any difference atthe various reducing reagents in MI94splicing process.The effectof tempera- ture and p H on MI94cleavage is more obvious than thatof MI84.In addition,a splicing mechanism ofthese mutants was discussed.

关 键 词：剪切 内蛋白子 突变体 DNA聚合酶 蛋白质自剪接 

分 类 号：Q78[生物学—分子生物学]