Development of rapid proteomic analysis for effective production of recombinant proteins by secretion

由于生物活性蛋白质通过容易的分离方法获得，因此重组蛋白质已经通过从重组细胞分泌来产生。然而，由于信号序列的不正确加工和蛋白酶的降解，靶蛋白的生产率并不总是高的。本研究以α-淀粉酶为模型蛋白，通过蛋白质组学分析，对重组蛋白进行了快速鉴定，并对重组蛋白中的标志酶和蛋白酶的活性进行了测定，提出了一种有效的重组蛋白分泌控制和生产方法。酿酒酵母和毕赤酵母的发酵条件，研究了发酵条件对菌体产量、标志酶活性及分泌蛋白特性的影响。获得以下结果：* 通过使用针对C-末端序列的抗肽抗体来阐明分泌的α-淀粉酶的C-末端的特征。*甲醇诱导毕赤酵母能有效地分泌α-淀粉酶，甲醇的补料量可以通过DO-statt来控制。开发了一种利用免疫脂质体的新型膜印迹方法，用于快速和高灵敏度的蛋白质测量。甲醇诱导提高了甲醇代谢途径中酶的活性，并与α-淀粉酶的比产率有关。

Recombinant proteins have been produced by secretion from recombinant cells, since biologically active proteins are obtained through easy separation processes. However, the productivity of target proteins is not always high because of incorrect processing of signal sequences and degradation by proteases. In this work we propose an effective control and production method for secretion of recombinant proteins based on the proteomic analysis with rapid characterization of produced proteins and measurements of the activities of marker enzymes and proteases.As a model protein α-amylase was secreted from S. cerevisiae and Pichia pastoris, and the effects of fermentation conditions on the productivity, the activities of marker enzymes and the characteristics of secreted protein were studied. The following results were obtained;* Characteristics of C-terminus of secreted α-amylase were clarified by use of an anti-peptide antibody against the C-terminal sequences.* Pichia pastoris could effectively secrete α-amylase by induction with methanol, and the feed of methanol could be controlled by the DO-statt for effective production.* A novel membrane blotting method utilizing immunoliposomes was developed for rapid and highly sensitive measurement of proteins.* The activities of enzymes in the metabolic pathway of methanol increased by induction with methanol and were related to the specific production rate of α-amylase.