运用翻译起始区结构优化实现广谱抗病毒蛋白PA在大肠杆菌中的高效表达  

Efficient Expression of the Broad-spectrum Antiviral Protein PA in Escherichia coli by Using Structural Optimization of the Translation Initiation Region

作　　者：李秋烨 谢德健 史姝 蔡秀怡 谭志霞[1,3] 张彦 沈文龙 叶湘漓 赵志虎[2] LI Qiuye;XIE Dejian;SHI Shu;CAI Xiuyi;TAN Zhixia;ZHANG Yan;SHEN Wenlong;YE Xiangli;ZHAO Zhihu(School of Medicine,Hunan Normal University,Changsha 410013,China;Laboratory of Advanced Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China;The Ninth Hospital of Changsha,Changsha 410004,China)

机构地区：[1]湖南师范大学医学院,长沙410013 [2]军事科学院军事医学研究院前沿生物技术实验室,北京100071 [3]长沙市第九医院,长沙410004

出　　处：《中国生物工程杂志》2024年第9期53-63,共11页China Biotechnology

基　　金：国家科技重大专项(2018ZX10305410);国家自然科学基金(31370762);湖南省卫生健康委员会重点项目(202201065690)资助项目。

摘　　要：大肠杆菌是重要的生物工程宿主系统,约1/3上市蛋白质类生物技术药物由大肠杆菌重组生产。但是,密码子偏性所致目的蛋白表达水平较低、不具备真核生物部分翻译后修饰等多种因素,限制了大肠杆菌系统的广泛应用。广谱抗病毒蛋白,对类似新冠的新突发再发传染性疾病的高效防控具有重要意义。为了实现广谱抗病毒蛋白PA在大肠杆菌中的高效表达、拓展其应用潜能,通过密码子优化、结合mRNA翻译起始区(translation initiation region,TIR)二级结构最小自由能(minimum free energy,MFE)预测等,对PA在大肠杆菌中的表达水平进行了研究。结果发现:密码子的优化,可以实现PA在大肠杆菌中的表达,但是总体表达水平并不是十分理想。TIR二级结构MFE的大小与表达水平高低密切相关,通过密码子的同义突变进而改变TIR局部二级结构、MFE大小,可以进一步提高或降低PA的表达水平,从而从正反两个方面证实全局密码子的优化结合TIR局部二级结构驱动的MFE调整,是实现PA在大肠杆菌中高效表达的一种现实、有效策略,对蛋白质类生物技术药物在原核系统中的高效表达与规模化生产,具有重要应用价值。Escherichia coli is an important bioengineering host system,and about 1/3 of the marketed protein-based biotech drugs are produced by E.coli.However,many factors such as low expression levels of target proteins due to codon bias and the lack of partial post-translational modifications in eukaryotes limit the widespread use of the E.coli system.The broad-spectrum anti-viral protein is of great significance for the effective prevention and control of outbreaks of emerging and re-emerging infectious diseases similar to the new corona virus.In order to achieve the efficient expression of the broad-spectrum anti-virus protein PA in E.coli and expand its potential clinical applications,the expression level of PA in E.coli was studied by codon optimization and prediction of the minimum free energy(MFE)of the secondary structure of the translation initiation region(TIR)of the mRNA.The results showed that codon optimization can partially achieve high expression of PA in E.coli,but the overall expression level was not very satisfactory.We found that the MFE value of the TIR’s secondary structure of the TIR of our chosen window is closely positively related to the level of expression.Synonymous mutation of codons can alter the local secondary structure of TIR and the value of MFE,which can be further used to increase or decrease the expression level of PA.From both positive and negative aspects,it is confirmed that the global codon optimization combined with local adaptation of the MFE value of TIR driven by the local secondary structure of TIR is a realistic and effective strategy to achieve the efficient expression of PA in E.coli.This approach has important application value for the large-scale production of protein biotech drugs in the prokaryotic system.

关 键 词：大肠杆菌 原核表达 翻译起始区 MRNA二级结构 密码子优化 

分 类 号：Q819[生物学—生物工程]