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启动子改造及发酵条件优化提高人源III型胶原蛋白在毕赤酵母中的表达量

发表时间:2020-10-10  浏览量:439  下载量:70
全部作者: 刘超,张萌,许菲
作者单位: 江南大学生物工程学院
摘 要: 通过微生物异源表达胶原蛋白能够避免传统提取方法存在的有致病隐患和水溶性低的问题,也能突破化学合成方法对胶原蛋白长度的限制,使得对胶原序列的设计更加灵活。本文合成了截短的人源III型胶原蛋白N-HC3240基因,为提高其在毕赤酵母中的表达量,通过启动子改造策略,研究了启动子AOX1、DAS1、DAS2、FLD1、FDH1、THI4和THI11对N-HC3240在毕赤酵母GS115中表达的影响,结果表明AOX1启动子是N-HC3240时空表达量最优的启动子。此外,对AOX1启动子的启动表达,进行了溶氧、初始诱导菌体浓度、诱导pH值和甲醇浓度的优化,最终N-HC3240在摇瓶水平的产量达到0.28 g/L,是优化前的2.5倍,并通过质谱鉴定了N-HC3240的序列。本研究为人源III型胶原蛋白在毕赤酵母中的异源高效表达提供了一定的研究基础。
关 键 词: 生物工程;胶原蛋白;毕赤酵母GS115;启动子;发酵优化
Title: Promoter modification and fermentation condition optimization enhance the expression of human type III collagen in Pichia pastoris
Author: LIU Chao, ZHANG Meng, XU Fei
Organization: School of Biotechnology, Jiangnan University
Abstract: The heterologous expression of collagen by microorganisms can not only avoid the hidden dangers of traditional extraction methods and low water solubility, but also break through the restrictions on the length of collagen in chemical synthesis methods, making the design of collagen sequence more flexible. In this paper, a truncated human type III collagen N-HC3240 gene was synthesized, in order to increase its expression in Pichia pastoris, the effects of promoters AOX1, DAS1, DAS2, FLD1, FDH1, THI4 and THI11 on the expression of N-HC3240 in P. pastoris GS115 were studied by promoter modification strategy. The results showed that AOX1 promoter was the best promoter for N-HC3240 space-time yield. And the expression of N-HC3240 using AOX1 promoter was optimized by dissolved oxygen, initial induced bacterial concentration, induced pH value and methanol concentration. Finally, the yield of N-HC3240 in shaking flask reached 0.28 g/L, which was 2.5 times of that before optimization. The correct expression of N-HC3240 was identified by mass spectrometry. This study provides a basis for the heterologous and high-level expression of human type III collagen in P. pastoris.
Key words: biotechnology; collagen; Pichia pastoris GS115; promoter; fermentation optimization
发表期数: 2020年9月第3期
引用格式: 刘超,张萌,许菲. 启动子改造及发酵条件优化提高人源III型胶原蛋白在毕赤酵母中的表达量[J]. 中国科技论文在线精品论文,2020,13(3):292-302.
 
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