基于3A组装的多拷贝基因策略优化重组水蛭素变体Ⅲ的生产  

Efficient production of Rhv3 using a multi-copy gene strategy based on 3A assembly

作　　者：王亚丽 刘秀霞 杨艳坤[1,2,3] 白仲虎 WANG Yali;LIU Xiuxia;YANG Yankun;BAI Zhonghu(National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214112,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学,粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122 [2]江南大学,工业生物技术教育部重点实验室,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2023年第16期1-8,共8页Food and Fermentation Industries

基　　金：国家自然科学基金(21878124,22078128,21938004)。

摘　　要：水蛭素变体Ⅲ(Hirudin varidantⅢ,Hv3)是一种从水蛭中提取的活性成分,在预防和治疗白内障方面具有潜在作用。为了制备足量Hv3用于进一步的临床前应用研究,该研究开发了一种在谷氨酸棒杆菌(Corynebacterium glutamicum)中高效、稳定表达重组水蛭素变体Ⅲ(recombinant Hirudin varidantⅢ,Rhv3)的办法。首先,在C.glutamicum中构建含Ptac启动子和CspA信号肽的Rhv3分泌表达菌株,比较其在不同培养基中的表达情况。结果表明在BHI培养基中Rhv3活性最高,达到3.02×10^(3) ATU/L。为进一步提升Rhv3产量,通过核糖体结合位点(ribosome binding site,RBS)筛选,选用RBS1应用于Rhv3多拷贝菌株构建和表达。然后利用3A组装技术构建含不同信号肽和不同拷贝数的Rhv3分泌表达菌株进一步优化其产量。通过放大发酵培养,Rhv3产量达到1.89 g/L,活性达到10.91×10^(3) ATU/L。最后利用镍柱对Rhv3进行简单纯化,其纯度达到90%。该异源表达策略有效提高Rhv3表达量,为Rhv3的重组生产提供了一种质量可靠、低成本的表达体系。Hirudin variantⅢ(Rhv3),a leech derived active ingredient,has the potential effect on the prevention and treatment of cataracts.To prepare sufficient amounts of Rhv3 for clinical research,the effective and stable process for Rhv3 expression in Corynebacterium glutamicum was developed in this study.Firstly,Rhv3 secretion expression strain,which was controlled by Ptac promoter and CspA signal peptide,was constructed in C.glutamicum.The expression level of this strain was compared in different media.The results showed that Rhv3 activity was highest in the BHI medium and reached 3.02×10^(3) ATU/L.Next,the RBSs was screened using egfp reporter gene to increase production of recombinant protein.And the RBS1 was finally selected and applied to the construction and expression of the strain of multi-copy strain of Rhv3.By screening the signal peptide and copy number using 3A assembly,the six was selected as the best copy number of Rhv3.It was found that this strain could promote the expression level to the maximum extent in C.glutamicum.Further increased production of Rhv3 was achieved in large-scale fermentation,the M6S-PorB strain was used to produce Rhv3.Its yield was approximately accumulate 1.89 g/L,and the activity of Rhv3 reached 10.91×10^(3) ATU/L,which was 3.61 times higher than that of the starting strain.Finally,the Rhv3 was purified by nickel column,and the purity was higher than 90%.To the best of our knowledge,this is the first report of multi-copy strategy to produce recombinant protein in C.glutamicum.This strategy based on the 3A assembly would provide an expression system with effective,reliable,and low cost for recombinant protein expression in C.glutamicum.

关 键 词：重组水蛭素变体Ⅲ(recombinant Hirudin varidantⅢ Rhv3) 核糖体结合位点(ribosome binding site RBS) 多拷贝 信号肽 谷氨酸棒杆菌 

分 类 号：Q789[生物学—分子生物学] TQ920.1[轻工技术与工程—发酵工程]