法夫驹形氏酵母无细胞蛋白合成系统的条件优化

On the Optimization of Cell-Free Protein Synthesis of Komagataella phaffii

无细胞蛋白质表达系统在重组蛋白表达、基因表达调控应用、生物检测等方面具有独特优势。文章建立了利用法夫驹形氏酵母细胞裂解物、包含内部核糖体进入位点、T7启动子和poly(A)重组质粒的无细胞蛋白质表达系统,分别优化了该系统中7个组分对绿色荧光蛋白表达的影响,进而利用DSD响应面优化得到该系统的最佳组分浓度,使绿色荧光蛋白的荧光强度增加了3.5倍。该研究结果为法夫驹形氏酵母无细胞系统的进一步应用奠定了理论与数据基础。

Cell-free protein synthesis system has unique advantages in recombinant protein expression,gene regulation,bio-detection.In this study,Komagataella phaffii based cell-free protein synthesis system was established using recombinant plasmids containing internal ribosome entry sites,T7 promoter and poly(A).The effects of seven components on the expression of green fluorescent protein by cell-free protein synthesis system were optimized.And then,DSD response surface optimization was used to obtain the optimal component concentration of cell-free protein synthesis system.The fluorescence intensity of eGFP was increased by 3.5 fold after optimization systematically.The results built a solid foundation for the further application of Komagataella phaffii cellfree protein synthesis system.