快速筛选高效表达重组蛋白毕赤酵母菌株新方法的建立及评价

Development and evaluation of a novel method for rapid screening of Pichia pastoris strains capable of efficiently expressing recombinant proteins

摘要:

毕赤酵母是当前应用最为广泛的重组蛋白表达系统之一，文中建立了一种快速筛选高效表达重组蛋白的毕赤酵母菌株的新方法。首先，对内质网转膜蛋白Sec63融合表达增强型绿色荧光蛋白EGFP的改造菌株GS115-E表达重组蛋白的能力进行检测；之后将携带不同拷贝数的植酸酶phy基因或木聚糖酶xyn基因的质粒转化进入GS115-E中，得到具有不同植酸酶或木聚糖酶表达水平的重组菌株，分别检测不同菌株的EGFP与重组蛋白的表达水平；最后，利用分选型流式细胞仪，根据绿色荧光值的高低对包含不同植酸酶表达水平的重组菌株的菌群进行分选。结果显示重组菌株中EGFP的荧光值与重组蛋白的活性表达水平之间具有良好的线性相关性(0.8<|R|<1)，且利用流式细胞仪可高效地从混合菌群中筛选得到高产菌株，所分选得到的高荧光菌株在摇瓶发酵120 h时植酸酶表达水平是低荧光菌株的4.09倍。本方法通过检测菌株的EGFP荧光值代替检测重组蛋白的表达水平和活性，从而实现高表达菌株的筛选，大大提高了其应用的便捷性及通用性。与流式细胞仪、液滴微流控等高通量筛选仪器或技术结合将进一步提高筛选的速度与通量，为筛选获得高效表达重组蛋白的毕赤酵母菌株提供了简便、快速的新途径。

Abstract:

Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.