CAJ | 학술논문

为获得 ALAS的重组表达蛋白,以人肝脏组织 cDNA为模板,通过 PCR扩增得到 ALAS全长片段并测序,成功构建了 pET30a(+)—ALAS融合表达载体并转化大肠杆菌 Rosetta (DE).IPTG诱导融合蛋白表达. SDS变性凝胶电泳结果显示,融合蛋白的分子量约70 kDa,并且在上清液中有少量表达.经镍柱一步纯化得到His6—ALAS融合蛋白.
위획득 ALAS적중조표체단백,이인간장조직 cDNA위모판,통과 PCR확증득도 ALAS전장편단병측서,성공구건료 pET30a(+)—ALAS융합표체재체병전화대장간균 Rosetta (DE).IPTG유도융합단백표체. SDS변성응효전영결과현시,융합단백적분자량약70 kDa,병차재상청액중유소량표체.경얼주일보순화득도His6—ALAS융합단백.
To express and purifyδ—aminolevulinic acid (ALAS)in E.coli,liver RNA was reverse tran—scribed into cDNA.The ALAS gene was amplified by PCR and confirmed by sequencing.The pET30a(+)—ALAS recombinant vector was successfully constructed.The recombinant vector was transformed into Rosetta (DE3 )and induced by IPTG.SDS—PAGE analysis showed that the fusion protein was about 70 kDa,and a small portion of the protein was expressed in the supernatant.The expressed His6—tagged pro—tein was purified by Ni2+ affinity chromatography column and characterized by SDS—PAGE and confirmed by western blot analysis.