苦豆子SaLDC密码子偏好性分析、优化及原核表达

目的 研究苦豆子SaLDC密码子偏好性、优化及原核表达.方法 运用EMBOSS在线程序中的CHIPS、CUSP和CodonW软件分析SaLDC密码子的偏好性,根据大肠杆菌密码子的偏好性对SaLDC密码子进行优化,并将优化后的基因命名为optSaLDC;构建重组表达载体pET-28a (+) -optSaLDC,转化大肠...

Codon usage bias analysis,optimization and prokaryotic expression of SaLDC in Sophora alopecuroide

AIM To analyze the codon usage bias,optimization and prokaryotic expression of the lysine decarboxylase gene of Sophora alopecuroides(SaLDC).METHODS With CHIPS,CUSP and CodonW module of EMBOSS online program,the codon usage bias of SaLDC was analyzed.The codons of SaLDC were further optimized with reference to the codon usage bias of E.coli.The optimized gene was named optSaLDC.The recombinant expression vector pET?28a(+)?optSaLDC was constructed with the target gene optSaLDC and pET?28a(+)vector and then genetically transformed into E.coli BL21(DE3).The expression of IPTG?induced SaLDC was analyzed using mass spectrometry.RESULTS The relative synonymous codon usage(RSCU)of 28 codons in SaLDC were larger than 1.00,and the preference was shown with synonymous codons ending with A or T.The codon usage bias of SaLDC was the closest to that of Arabidopsis thaliana,and the furthest away from that of E.coli.After optimization,the optimum expression condition of optSaLDC in E.coli was 1.0 mmol/L IPTG at 15℃for 16 h,and the molecular weight of the target fusion protein was measured to be 48990.51 Da by LC?MS/MS.CONCLUSION The purified IPTG?induced optSaLDC protein can be used to study in vitro enzyme activity of SaLDC,and its more function as well.