CAJ | 학술논문

为了提高质粒pHsh-celD在大肠杆菌中的表达量,运用 mRNA 二级结构预测软件对pHsh-celD翻译起始区的二级结构进行优化,得到了具有最佳 mRNA二级结构及自由能的质粒pHsh-celD I。为了进一步提高纤维素酶的表达量,在不改变目的基因翻译的氨基酸序列的前提下,celD的N-端氨基酸尽可能地使用大肠杆菌同义密码子,定点突变后得到质粒pHsh-celD II。优化后质粒pHsh-celD II表达的纤维素酶活[(6.4±0.4) U/ml]是未优化[(4.1±0.3) U/ml]的1.6倍。 SDS-PAGE 结果显示,该重组酶的分子量为66000,与理论值相符。基于热激载体pHsh 的重组表达系统具有诱导表达简便、诱导方式廉价的优点,且重组酶热稳定性好,这对该酶的大规模发酵应用具有重要意义。
위료제고질립pHsh-celD재대장간균중적표체량,운용 mRNA 이급결구예측연건대pHsh-celD번역기시구적이급결구진행우화,득도료구유최가 mRNA이급결구급자유능적질립pHsh-celD I。위료진일보제고섬유소매적표체량,재불개변목적기인번역적안기산서렬적전제하,celD적N-단안기산진가능지사용대장간균동의밀마자,정점돌변후득도질립pHsh-celD II。우화후질립pHsh-celD II표체적섬유소매활[(6.4±0.4) U/ml]시미우화[(4.1±0.3) U/ml]적1.6배。 SDS-PAGE 결과현시,해중조매적분자량위66000,여이론치상부。기우열격재체pHsh 적중조표체계통구유유도표체간편、유도방식렴개적우점,차중조매열은정성호,저대해매적대규모발효응용구유중요의의。
In order to improve the expression level of celD gene, through structural optimization of plasmid pHsh-celD, the plasmid pHsh-celD I, which possessed the most appropriate structure and free energy of mRNA, was ob-tained. Higher expression level was achieved by replacing the rare codons for N-terminal amino acids of the target protein, and the ultimate plasmid, pHsh-celD II was resulted. The expression level of celD was increased from (4. 1±0. 3) U/ml to (6. 4±0. 4) U/ml in LB medium. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant celD was about 66 000, exactly the size predicted. The expression vector system of the heat shock plasmid pHsh owned advanta-ges such as high expression level and cheap induction. Moreover, the superior stability of the recombinant enzyme laid the base for the application of large-scale fermentation.