CAJ | 학술논문

通过酶切法从重组质粒pUC18-chnB中得到编码环己酮单加氧酶的chnB基因序列,将其定向插入原核表达载体pQE30中,构建重组质粒pQE30-chnB,转化到大肠杆菌BL21中并诱导表达目的蛋白,用SDS-PAGE电泳检测表达产物.测序结果表明chnB基因大小为1 623bp,编码由540个氨基酸残基构成的多肽.SDS-PAGE结果显示,表达分子量约为60kDa的带有6×His标签的环己酮单加氧酶,与预期分子量相符,表明成功构建出原核表达质粒并实现了目的蛋白表达,为进一步开展环己酮单加氧酶活性研究奠定了基础.
통과매절법종중조질립pUC18-chnB중득도편마배기동단가양매적chnB기인서렬,장기정향삽입원핵표체재체pQE30중,구건중조질립pQE30-chnB,전화도대장간균BL21중병유도표체목적단백,용SDS-PAGE전영검측표체산물.측서결과표명chnB기인대소위1 623bp,편마유540개안기산잔기구성적다태.SDS-PAGE결과현시,표체분자량약위60kDa적대유6×His표첨적배기동단가양매,여예기분자량상부,표명성공구건출원핵표체질립병실현료목적단백표체,위진일보개전배기동단가양매활성연구전정료기출.
The chnB gene encoding a cyclohexanone monooxygenase was isolated from the recombinant plasmid pMD18-chnB by digested with BamHI and HindⅢ,then cloned into the expression vector pQE30 and expressed in E.coli BL21 by IPTG induction.The expressed protein was identified by SDS-PAGE.The sequence analyses showed that the coding region of the chnB of Rhodococcus ruber JDM312 was 1 623 bp in length,and the predicted primary protein was composed of 540 amino acids.The SDS-PAGE analyses revealed a single protein band with a molecular weight of 60 kDa,suggesting that the recombinant plasmid of pQE30-chnB was successfully constructed and the target protein was expressed in E.coli.