不同标签对致病疫霉PiGK5胞外端表达及稳定性的影响

Effects of Different Tags on the Expression and Stability of Extracellular Terminal of Phytophthora infestans PiGK5

摘要: 马铃薯晚疫病病原菌致病疫霉的PiGK5蛋白是一类跨膜蛋白，该蛋白在致病疫霉的致病性、无性生殖及有性生殖方面均有重要作用，但其结构与详细功能目前尚未可知。本研究通过PCR扩增PiGK5胞外端编码区，分别构建融合His标签及MBP和His双标签的原核表达载体，比较了不同标签对PiGK5胞外端表达水平、可溶性及稳定性的影响。结果显示，融合His标签的PiGK5胞外端蛋白主要以包涵体形式存在于沉淀中，仅有少量以可溶性蛋白的形式存在，1 L大肠杆菌表达的蛋白总量为28.11 mg，其中可溶性蛋白总量约为2.73 mg，而纯化后可获得纯蛋白量为0.1175 mg。纯化后的重组蛋白稳定性较好，可在96小时内维持蛋白结构稳定。融合MBP、His双标签的PiGK5胞外端蛋白主要以可溶性蛋白的形式存在，1 L大肠杆菌表达的蛋白总量为49.93 mg，其中可溶性蛋白总量约为44.73 mg，而纯化后可获得纯蛋白量为6.0125 mg。但重组蛋白的稳定性较差，仅在1.5小时就出现了蛋白降解现象。因此，虽然融合His标签的PiGK5胞外端蛋白的可溶性蛋白量较低，但重组蛋白更稳定，融合His标签更适合于PiGK5胞外端的原核表达及后续结构与功能研究。本研究比较了不同标签对PiGK5蛋白胞外端表达的影响，建立了该蛋白的原核表达条件，为其结构与功能的研究奠定了基础。

Abstract: PiGK5 protein of potato late blight pathogen Phytophthora infestans is a kind of transmembrane protein, which plays an important role in pathogenicity, asexual reproduction and sexual reproduction of P. infestans. However, the structure and detailed function of this protein have not been known. In this study, the coding region of cDNA of extracellular terminal of PiGK5 was amplified by PCR, and the prokaryotic expression vectors fused with His tag and fused with MBP and His tags were constructed respectively. The effects of different tags on the expression level, solubility and stability of extracellular terminal of PiGK5 were compared. The results showed that the extracellular terminal of PiGK5 fused with His tag mainly existed in the form of inclusion body, and only a small amount of protein was soluble. The total amount of the protein expressed in 1 L culture medium of Escherichia coli was 28.11 mg, in which the total amount of soluble protein was about 2.73 mg, while the amount of the protein obtained after purification was 0.1175 mg. The purified recombinant protein was stable and could maintain structural stability within 96 hours. The extracellular terminal of PiGK5 fused with MBP and His double tags mainly existed in the form of soluble protein. The total amount of the protein expressed in 1 L culture medium of E. coli was 49.93 mg, in which the total amount of soluble protein was about 44.73 mg, while the amount of the protein obtained after purification was 6.0125 mg. However, the stability of the recombinant protein was poor, and the protein degradation appeared only in 1.5 hours. Therefore, although the soluble protein content of PiGK5 extracellular terminal fused with His tag is lower, the recombinant protein is more stable and suitable for prokaryotic expression and follow-up study of structure and function of extracellular terminal of PiGK5. In this study, the effects of different tags on the expression of PiGK5 extracellular terminal were compared, and the prokaryotic expression conditions of the protein were established, which laid a foundation for the study of its structure and function.