培养温度对多拷贝毕赤酵母猪胰岛素前体的分泌表达和生理特性影响

Effect of Temperature on the Secretory Expression of Porcine Insulin Precursor and Physiological Characterization of Multicopy P. pastoris

摘要: 研究了培养温度对多拷贝毕赤酵母分泌表达猪胰岛素前体（PIP）的生理特性的影响。将多拷贝重组菌A2（12拷贝）和A3（18拷贝）分别在25 ℃和 30 ℃培养，25 ℃培养时PIP表达水平分别比30 ℃培养时提高了40%和32%，而A3细胞的存活率提高了20%。对A3细胞内相关蛋白折叠和氧化程度的KAR2，PDI1, GLR和TRR1基因转录水平分析发现，相比30 ℃培养25 ℃培养时减少了16%~35%，这表明多拷贝重组A3菌株在低温培养时比高温培养在蛋白折叠和氧化协廹响应有明显的减缓效应，从而导致外源蛋白表达提高了40%.

Abstract: The impact of cultivation temperature on the physiology and productivity of porcine insulin precursor (PIP) protein secreting P. pastoris strains was investigated. The multicopy PIP producing P. pastoris strains A2 and A3 were cultivated at 25 ℃ and 30 ℃. Compared with those at 30 ℃， PIP production of A2 and A3 at 25 ℃ was increased by 40% and 32%, respectively. The viability of recombinant A3 cells was increased by 20%, and the transcriptional levels of KAR2, PDI1, GLR, and TRR1 in A3 cells were reduced by 16%-35% compared with those cultivated at 30 ℃. Thus, protein folding stress and oxidative stress responses in the highcopy strain cells are generally decreased at 25 ℃, leading to a 40% increase in heterologous protein production by multicopy recombinart P. pastoris host cells.