西北民族大学学报:自然科学版
西北民族大學學報:自然科學版
서북민족대학학보:자연과학판
Journal of Northwest Minorities University:Natural Science Edition
2011年
3期
54-57
,共4页
王冬梅%董开忠%蔡勇%刘翊中%杨具田
王鼕梅%董開忠%蔡勇%劉翊中%楊具田
왕동매%동개충%채용%류익중%양구전
ApxⅢ包涵体%纯化%变性
ApxⅢ包涵體%純化%變性
ApxⅢ포함체%순화%변성
ApxⅢ inclusion body%purification%denaturation
为摸索重组ApxⅢ蛋白的纯化条件,采用IPTG诱导pET~apxⅢA/JM109,表达产物以包涵体形式存在.收集诱导后菌体,经冻融-超声破菌得到粗包涵体,经10 mmol/L Tris—HCl、TritonX~100和2mol/L尿素依次洗涤.包涵体沉淀分别用含有8mol/L尿素和6mol/L盐酸胍的溶液变性溶解.聚丙酰胺凝胶电泳(SDS—PAGE)分析结果表明:经洗涤包涵体纯度提高;8mol/L尿素和6mol/L盐酸胍都不能使ApxⅢ包涵体完全溶解.
為摸索重組ApxⅢ蛋白的純化條件,採用IPTG誘導pET~apxⅢA/JM109,錶達產物以包涵體形式存在.收集誘導後菌體,經凍融-超聲破菌得到粗包涵體,經10 mmol/L Tris—HCl、TritonX~100和2mol/L尿素依次洗滌.包涵體沉澱分彆用含有8mol/L尿素和6mol/L鹽痠胍的溶液變性溶解.聚丙酰胺凝膠電泳(SDS—PAGE)分析結果錶明:經洗滌包涵體純度提高;8mol/L尿素和6mol/L鹽痠胍都不能使ApxⅢ包涵體完全溶解.
위모색중조ApxⅢ단백적순화조건,채용IPTG유도pET~apxⅢA/JM109,표체산물이포함체형식존재.수집유도후균체,경동융-초성파균득도조포함체,경10 mmol/L Tris—HCl、TritonX~100화2mol/L뇨소의차세조.포함체침정분별용함유8mol/L뇨소화6mol/L염산고적용액변성용해.취병선알응효전영(SDS—PAGE)분석결과표명:경세조포함체순도제고;8mol/L뇨소화6mol/L염산고도불능사ApxⅢ포함체완전용해.
Recombinant ApxⅢ was expressed in E. coli under IPTG induction. It existed as inclusion body. Cells were harvested, freeze - thawed, sonicated and centrifuged after expression, and the pellet (crude ApxⅢ inclusion body) was washed by successive steps as follows: 10 mmol/LTris- HC1, 1% TritonX- 100 and 2mol/L Urea. The precipitate was resolved and denatured by 8 mol/L Urea and 6 mol/ L Guanidine Hydrochloride respectively. The results of SAS - PAGE analysis show that purity of ApxⅢ inclusion body is improved by washed. The ApxⅢ inclusion body could not be dissolved completely in 8mol/L Urea and 6mol/L Guanidine Hydrochloride.