文献类型：期刊文献

中文题名：溶组织梭菌胶原酶H在大肠杆菌中的分泌表达

英文题名：Secretory Expression of Clostridium histolyticum Collagenase H in Escherichia coli

作者：赵钱山[1];刘晓[2];李素霞[1]

机构：[1]华东理工大学生物反应器国家重点实验室,上海200237;[2]上海雅心生物技术有限公司,上海200231

年份：2022

卷号：48

期号：6

起止页码：797

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：Scopus;北大核心:【北大核心2020】；CSCD:【CSCD_E2021_2022】；

语种：中文

中文关键词：溶组织梭菌胶原酶H;正交实验;信号肽;镁离子;分泌表达

外文关键词：Clostridium histolyticum collagenase H;orthogonal experiment;signal peptide;magnesium ion;secretory expression

摘要：溶组织梭菌Clostridium histolyticum胶原酶H(ColH)作用于胶原蛋白的Y-Gly处,将其水解成小分子肽。通过在colH基因中融合大肠杆菌外膜蛋白A的信号肽序列,成功构建了分泌高分子量的ColH(116 kDa)的菌株,结果发现信号肽在N端的位置影响其分泌效果,N端若有多余的氨基酸片段会显著降低信号肽引导胶原酶的分泌功能。通过正交试验与单因素实验对诱导条件和培养基添加剂优化提高其分泌表达量,结果表明:在诱导温度为25℃、诱导菌浓OD=0.9、IPTG(异丙基-β-D-硫代吡喃半乳糖苷)浓度为0.1 mmol/L、装液量为20%,镁离子浓度为10 mmol/L,诱导2.5 h后添加w为2%甘氨酸、诱导培养20 h,胞外胶原酶活性最高为0.68 U/mL,是优化前酶活的38.1倍,显著提高了分泌表达量。研究结果还表明,镁离子的添加可增加ColH的胞外分泌。
Clostridium histolyticum collagenase H(ColH) recognizes the Y-Gly of collagen and hydrolyzes it into small peptides. The high molecular weight ColH(116 kDa) secreting strain was successfully constructed by fusing colH gene with signal peptide sequence of outer membrane protein A. In this study, we found that the secretion of ColH was affected by the position of the signal peptide at the N-terminal, and the presence of excess amino acid fragments at the N-terminal significantly reduced the secretion function of the signal peptide-guided collagenase. Orthogonal experiment and single factor experiment were used to optimize the induction conditions and medium additives to improve the secretory expression. Under the conditions of inducing temperature of 25 ℃, the cell density(OD) of 0.9, IPTG concentration of 0.1 mmol/L, liquid volume of 20%, magnesium ion concentration of 10 mmol/L, and 2% glycine were added at 2.5 h after induction, the highest extracellular collagenase activity was 0.68 U/mL after induction for 20 h,which was 38.1 times of that of before optimization, and the secretory expression level was remarkably increased.Glycine added into the culture medium is a common strategy to promote the secretion of recombinant protein.Experimental results demonstrated that the amount and time of glycine added after induction showed the greatest influence on the secretion of collagenase. The addition of calcium and magnesium ions in the medium can promote the growth of E.coli. The results also showed that only the addition of magnesium ion can promote the secretion of ColH.