串联杂种泛素结合结构域蛋白（ThUBD）在大肠杆菌中的可溶性高效表达

目的：提高人工串联杂种泛素结合结构域蛋白（ tandem hybrid UBD， ThUBD）在大肠杆菌中的可溶性表达量，为泛素化蛋白研究提供高效特异的富集方法。方法根据大肠杆菌同义密码子相对频率（ RFSC）表对大肠杆菌的密码子进行分类，计算ThUBD密码子的相对适应度，并根据分析结果对ThUBD的密码子进行优化；诱导表达和蛋白定量检测密码子的优化结果；亲和纯化及泛素化蛋白富集研究检测密码子优化后的ThUBD－S的UbC结合能力。结果根据分析结果优化ThUBD密码子，使得适于大肠杆菌基因表达的密码子从48％增加到75％，并消除了阻碍转录翻译的密码子，其密码子适应指数（CAI）从0．63升高到0．88。密码子优化后的ThUBD其可溶性蛋白ThUBD－S的表达量增加了4倍，达到总蛋白的13．06％。同时，优化后的ThUBD－S可能具有更高的UbC的结合能力。结论 ThUBD的基因序列经过优化后，其在大肠杆菌中的表达量升高4倍左右。同时，序列优化不影响融合蛋白ThUBD在大肠杆菌中的可溶性表达和结合UbC的能力。

Soluble expression of tandem hybrid ubiquitin-binding domains (ThUBD) in prokaryotic cytoplasm of Escherichia coli BL21(DE3)

Objective We increase the soluble expression of artificial tandem hybrid ubiquitin binding domains ( ThUBD) in prokaryotic cytoplasm of Escherichia coli BL21 ( DE3 ) , which offer an effective and special profiling for ubiquitin conjugates( UbC) .Methods Codon optimization of the ThUBD was performed, followed by analysis of codon relative adaptiveness based on relative frequency of synonymous codon ( RFSC) of E.coli.Further induced expression and yeast ubiquitin conjugate enrichment quantified the soluble ThUBD-S and tested the ability to bind UbC.Results The statistical result showed that the percentage of codon of the highest usage frequency was increased from 48%to 75%, and codon adaptation index( CAI) was increased from 0.63 to 0.88 after codon optimization, which might suggest a higher expression of the ThUBD in E.coli BL21 (DE3).The subsequent SDS-PAGE indicated that the soluble target protein was increased four times, which accounted for 13.06%of total cell lysis.Further ubiquitinated proteome of yeast demonstrated that the ability to bind and enrich UbC of optimized ThUBD-S did not change compared with original ThUBD.Conclusion The expression of ThUBD-S can quadruple after codon optimization.At the same time, codon optimization does not impact its soluble expression and the ability to bind UbC.