人乳头瘤病毒16型L1基因在毕赤酵母中表达的影响因素分析

Impact Factors on the Expression of Recombinant Human Papillomavirus 16 L1 Protein in Pichia pastoris

目的:优化人乳头瘤病毒16型主要衣壳蛋白L1(human papillomavirus type 16 major capsid protein L1,HPV16L1)在毕赤酵母中的表达,并考察可能的影响因素。方法:四个不同序列特征的HPV16L1基因M16、Y16、P16、W16(其中,M16和Y16按酵母密码子优化,P16为哺乳动物细胞密码子优化,而W16为野生型序列)分别克隆于毕赤酵母表达质粒p PinkTM-HC(高基因拷贝菌落筛选)和p PinkTM-LC(低基因拷贝菌落筛选),并转化不同蛋白酶缺陷的宿主菌。甲醇诱导24小时后,取菌体样品经Western blot分析L1蛋白的表达。结果:M16显示了最高的表达水平,其次是Y16与P16,而W16几乎无表达。基因序列密码子应用特征分析显示,4个基因的密码子适应指数从高到低依次为Y16、M16、W16和P16。通过自由能和GC含量分析4个序列的mRNA二级结构,Y16为-409.40 kcal/mol和43.85%;M16为-451.50 kcal/mol和47.83%;P 16为-606.50kcal/mol and 64.10%;W16为-384.70 kcal/mol and 38.01%。蛋白酶缺陷菌株L1表达高于野生型菌株,质粒p PinkTM-HC与p PinkTM-LC介导的表达无明显区别。结论:密码子优化操作显著改善了HPV16L1在毕赤酵母中的表达,但表达水平与密码子利用优劣并不完全对应,提示密码子优化仅是部分原因,而mRNA结构与稳定性变化值得探讨。蛋白酶缺陷菌株提高了HPV16L1蛋白的稳定性,显著影响了表达水平。研究证明基因剂量对HPV16L1的表达未产生明显影响。

Optimization approaches have been shown in many published paper to be successful in improving the expression of a heterogenous gene in yeast cells,however,it is always needed to find out whether and how an approach works for the actual case. Multiple strategies were tried for the efficient expression of HPV16 L1 in Pichia pastoris,including the use of four gene versions with distinct sequence features（ M16 and Y16 optimized for Pichia pastoris cells,P16 for mammalian cells,and W16 from a wild type sequence,respectively）,and the employment of plasmid vectors facilitating selection of colonies with high or low gene copy numbers and host strains with different proteinase deficiency. M16 showed the highest expression,followed by Y16 and P16,whereas W16 was merely detectable. However,the codon adaptation order from high to low is sequentially Y16,M16,W16,and P16. The results indicate that the actually effective mechanism of codon optimization might not definitely attribute to codon adaptation. In addition,improving protein stability using specific proteinase-deficient host strains was also proved to be effective for L1 expression. In conclusion,the findings added useful information to our knowledge of optimizing expression of heterogeneous genes in Pichia pastoris cells.