Peng Meng, Tan Ming, Zeng Yan, Zheng Hongchen, Song Hui
Tianjin University of Science & Technology, Tianjin 300457, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Sheng Wu Gong Cheng Xue Bao. 2019 Jan 25;35(1):70-80. doi: 10.13345/j.cjb.180126.
Translocation ribonucleic acid (tRNA) is one of the important components in protein synthesis. In order to explore the effect of the changes of tRNAs corresponding to rare codons (rarity tRNAs) on the expression of exogenous genes, the co-expression system of rare tRNA gene and exogenous gene in Pichia pastoris was constructed. The expression of GFP in P. pastoris can be greatly reduced when a repressor region composed of four continuous proline rare codon CCG was added into the GFP gene. The expression amount of the repressed GFP could be increased about 4.9% when tRNAProCCG gene was cointegrated to the 3' of the repressed GFP gene through pPIC9K to the genome of P. pastoris GS115. Meanwhile, the expression amount of the repressed GFP increased about 12.5% by integrating the repressed GFP gene and tRNAProCCG gene to the genome of P. pastoris GS115 through pPIC9K and pFLDα, respectively. Using the same method, NFATc3T-GFP fusion gene and tRNAProCCG gene were co-expressed in P. pastoris GS115 resulting in 21.3% increased of the expression amount of NFATc3T-GFP fusion protein. In conclusion, tRNAProCCG gene has been confirmed to be a kind of rare tRNAs in P. pastoris GS115. Through co-expression of tRNAProCCG gene and heterologous genes which containing the continuous rare codon CCG, the expression of the repressed heterologous genes could be increased significantly. Furthermore, this co-expression system would contribute to screening and determining the other rare tRNAs.
转运核糖核酸（tRNA）是蛋白质合成中的重要组成部分之一。为了探究对应稀有密码子的tRNA（稀有tRNA）变化对外源基因表达的影响，构建了毕赤酵母中稀有tRNA基因与外源基因的共表达系统。当在绿色荧光蛋白（GFP）基因中添加由四个连续脯氨酸稀有密码子CCG组成的抑制区域时，毕赤酵母中GFP的表达会大幅降低。当通过pPIC9K将tRNAProCCG基因共整合到受抑制的GFP基因3'端至毕赤酵母GS115基因组时，受抑制的GFP表达量可提高约4.9%。同时，通过分别使用pPIC9K和pFLDα将受抑制的GFP基因和tRNAProCCG基因整合到毕赤酵母GS115基因组中，受抑制的GFP表达量提高了约12.5%。采用相同方法，在毕赤酵母GS115中共表达NFATc3T-GFP融合基因和tRNAProCCG基因，使得NFATc3T-GFP融合蛋白的表达量提高了21.3%。综上所述，已证实tRNAProCCG基因是毕赤酵母GS115中的一种稀有tRNA。通过tRNAProCCG基因与含有连续稀有密码子CCG的异源基因共表达，可显著提高受抑制的异源基因的表达。此外，该共表达系统将有助于筛选和确定其他稀有tRNA。
