Chen Yongan, Yuan Qingyan, Li Cheng, Liang Shuli, Lin Ying
School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Mar 25;37(3):939-949. doi: 10.13345/j.cjb.200629.
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.
毕赤酵母是应用最广泛的重组蛋白表达系统之一。在本研究中，开发了一种快速筛选能够高效表达重组蛋白的毕赤酵母菌株的新方法。首先，测试了修饰菌株GS115-E表达重组蛋白的能力，该菌株中功能性的Sec63-增强绿色荧光蛋白（EGFP）融合蛋白取代了内源性内质网跨膜蛋白Sec63。接下来，将携带不同拷贝数植酸酶（phy）基因或木聚糖酶（xyn）基因的质粒转化到GS115-E中，以获得具有不同植酸酶或木聚糖酶表达水平的重组菌株，并测试不同菌株中EGFP和重组蛋白的表达水平。最后，使用流式细胞仪分选器根据绿色荧光强度将具有不同植酸酶表达水平的细胞混合物分离成亚群。发现重组菌株中EGFP的荧光强度与重组蛋白的表达水平之间存在良好的线性相关性（0.8<|R|<1）。通过使用流式细胞仪，从细胞混合物中高效筛选出高产毕赤酵母细胞。在甲醇诱导120小时后，所选高荧光菌株的植酸酶表达水平比低荧光菌株高4.09倍。通过检测EGFP荧光强度而非检测重组菌株中重组蛋白的表达水平和活性，本研究开发的方法在筛选高产毕赤酵母菌株方面具有大大提高的便利性和通用性。将该方法与流式细胞仪和液滴微流控等高通量筛选仪器和技术相结合，该方法的速度和通量将进一步提高。该方法将为筛选和获得具有高重组蛋白表达能力的毕赤酵母提供一种简单快速的途径。
