真菌细胞色素P450在大肠杆菌中的表达

【背景】真菌细胞色素P450蛋白在大肠杆菌中表达水平低甚至不表达,近期研究发现通过对该类蛋白氨基端(N端)氨基酸序列的修饰可优化其表达水平。【目的】在大肠杆菌系统中表达预测功能为P450酶的焦曲霉094102菌株的Au8002蛋白,为真菌P450蛋白在大肠杆菌表达系统中的N端氨基酸序列修饰策略提供有效依据。【方法】对野生型P450蛋白Au8002的氨基酸序列进行分析,对其N端序列进行了3种序列修饰,并在诱导蛋白表达时添加P450生物合成前体5-氨基乙酰丙酸(5-ALA),研究N端氨基酸序列修饰策略及前体添加对真菌P450在大肠杆菌中蛋白表达的影响。【结果】SDS-PAGE和Westernblot检测结果显示,对目的蛋白进行的3种氨基酸序列修饰均使Au8002蛋白获得了表达,前体5-ALA的添加提高了目的蛋白表达量。其中对目的蛋白进行N端全长截短时可部分增加其可溶性,同时也验证了其特征性的CO结合能力。【结论】对预测为P450酶的菌株094102蛋白Au8002氨基端(N端)氨基酸序列的修饰有效解决了其在大肠杆菌内不表达的难题,实现了其可溶性表达;另一方面P450生物合成前体5-ALA的添加也能有效提高该类蛋白的表达水平,上述策略对改善其它该类蛋白在大肠杆菌内的表达水平具有借鉴意义。

Heterologous expression of a fungal cytochrome P450 in Escherichia coli

[Background] The expression of fugal P450s in Escherichia coli system is often in low efficiency. The N-terminal domain (NTD) amino acid sequence modification has been emerged as a potential strategy to achieve high-level expression of these membrane-bound proteins. [Objective] The Au8002 gene in Aspergillus ustus 094102 is predicted to encode a fungal P450. It cannot be expressed successfully in its natural sequence. Three NTD modifications were carried to prove the efficiency of this strategy on the heterologous expression in E. coli system. [Methods] Three NTD-modified Au8002 protein were designed to facilitate the gene expression and 5-aminolevulinic acid (5-ALA), the substrate for biosynthesis of P450s, was added during the heterologous expression to increase the yield of protein. [Results] The identifying results by SDS-PAGE and western blot indicated that the NTD modifications used in this study could improve the fungal gene expression and the addition of 5-ALA improved the P450 protein yield in E. coli. When the NTD was truncated in full length, the NTD-modified Au8002 appeared to be partially soluble, and CO binding test also proved its solubility and proved that it was a P450 protein. [Conclusion] The NTD modification of Au8002 of strain 094102 can help to overcome the low efficiency of its heterologous expression in E. coli and the addition of 5-ALA can also help to increase its expression level. The observation made in this study may provide a useful guideline for improving the expression yield of fungal P450s in E. coli.