文献类型：期刊文献

中文题名：赖氨酰内切酶在大肠杆菌中的重组表达、复性及纯化

英文题名：Recombinant Expression,Refolding,and Purification of Lysobacter enzymogenes Lys-C in Escherichia coli

作者：朱奕[1];徐名强[1];任燕娜[1];蔡孟浩[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2023

卷号：49

期号：5

起止页码：702

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_E2023_2024】；

语种：中文

中文关键词：赖氨酰内切酶;重组表达;复性;纯化;活性

外文关键词：lysyl endopeptidase;recombinant expression;renaturation;purification;activity

摘要：首先在产酶溶杆菌来源的赖氨酰内切酶(Lys-C)成熟肽序列的N端和C端分别融合人工前肽(MGSK)和6×His标签,将密码子优化后的序列插入表达载体pET-28a;其次以IPTG(异丙基硫代半乳糖苷)诱导型启动子PT7控制Lys-C的高效表达,并针对重组菌株JM109DE3_PT7-LysC开展生物反应器高密度发酵生产Lys-C;再次收集和溶解包涵体得到Lys-C变性液,通过Sephadex G25层析脱除DTT(二硫苏糖醇),并在Lys-C复性液中添加前导肽(pre-N-pro)辅助成熟肽蛋白折叠;进一步通过切向流过滤、Ni NTA-Sepharose亲和层析和Sephacryl S-100层析等一系列纯化步骤,获得高纯度的重组Lys-C;最后进行酶切三肽底物和门冬胰岛素前体检测,分析重组Lys-C的活性水平。结果表明:重组Lys-C的发酵产量为2.4 g/L,经复性和纯化后的终产量可达48 mg/L;添加80 mg/L pre-N-pro促进了重组Lys-C的复性,复性后酶活相比于未添加pre-N-pro时提升了4.8倍,最高可达到13.8 U/L;经过多步纯化后,重组Lys-C的比酶活为10.2 U/mg,且对于门冬胰岛素前体的酶切转化率可达93.5%。
This paper describes the development of a new recombinant expression and purification strategy for lysyl endopeptidase(Lys-C)in order to improve the efficiency of natural Lys-C production chain.An artificial pro-peptide(MGSK)and the 6×His tag were fused to the N-terminus and C-terminus of the Lys-C mature peptide gene sequence from Lysobacter enzymogenes,respectively,and then the codon-optimized sequence was inserted into plasmid pET-28a.Lys-C was efficiently expressed in the recombinant strain JM109DE3_P T7-LysC and controlled by the IPTG-inducible promoter PT7.The inclusion bodies were collected by high-density fermentation in a bioreactor,after which they were solubilized to obtain the Lys-C denaturing solution,and DTT was removed by Sephadex G25 chromatography.Then,the pre-pro peptide(pre-N-pro)was added to Lys-C refolding solution to assist in the folding of the mature protein.Further,the large volume Lys-C refolding solution was concentrated using tangential flow filtration,followed by a multi-step purification process including Ni NTA-Sepharose affinity chromatography,ultrafiltration,and Sephacryl S-100 chromatography to obtain high-purity recombinant Lys-C.Finally,the activity of recombinant Lys-C was measured by chromogenic reaction and digestion of insulin aspart precursors.The recombinant Lys-C fermentation yield was determined to be 2.4 g/L,and the final yield reached 48 mg/L after renaturation and purification.The enzymatic activity of Lys-C was improved by 4.8-fold(13.8 U/L)in the presence of 80 mg/L of pre-N-pro.The specific enzyme activity of Lys-C achieved 10.2 U/mg after multi-step purification,and the digestion efficiency of Lys-C for insulin precursors reached 93.5%.In this study,recombinant Lys-C with high yield and good activity was obtained allowing for accurate digestion of the insulin precursors,which provides an insight into Lys-C recombinant expression and potential industrial application.