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
首发时间:2020-06-15
摘要:胶原蛋白是人体细胞骨架和细胞外基质中的主要组成部分,作为生物材料其具有良好的生物相容性、低免疫性和生物可降解性等性质。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,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、初始诱导菌浓、诱导pH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。
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Promoter Modification and Fermentation Condition Optimization Enhance the Expression of Human Type III Collagen in Pichia pastoris
Abstract:Collagen is the most abundant component of human cytoskeleton and extracellular matrix. As a biomaterial, it has good biological properties, such as good biocompatibility, low immunity and biodegradability. 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. Through the strategy of promoter modification, the effects of promoters AOX1, DAS1, DAS2, FLD1, FDH1, THI4 and THI11 on the expression of N-HC3240 in Pichia pastoris were studied. The results showed that AOX1 promoter was the best promoter for N-HC3240 expression. And the expression of N-HC3240 using AOX1 promoter was optimized by dissolved oxygen, initial induced bacterial concentration, induced pH 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 Pichia pastoris.
Keywords: Collagen Pichia pastoris Heterologous expression Promotor Fermentation optimization
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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
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