酵母GS115中镍柱亲和层析结合蛋白的鉴定-生物化学与分子生物学专业论文.docxVIP

汕头大 汕头大学硕士学位论文 PAGE PAGE IV Abstract The importance of recombinant therapeutic protein production via heterologous expression systems has grown rapidly since the emergence of the modern biotechnology industry . The recent developments of heterologous expression systems include prokaryote , yeast, insect, mammalian cell and plants expression system. Among them, yeast expression system has the advantages of simple media requirements, rapid growth similar to prokaryotic system and eukaryotic ptotein processing like eukaryotic expression system. Yeast,as a unicellular eukaryote, offers a desired ease of genetic manipulation and rapid growth to high cell density on inexpensive media. It grows rapidly in large HYPERLINK /wiki/Fermenter fermenters, and produces proteins safely in an efficient way , and it is also performs protein modifications like HYPERLINK /wiki/Glycosylation glycosylation, thus producing even complex foreign proteins that are identical or very similar to native products from plant or mammalian sources. Yeast expression system mainly contains Saccharomyces cerevisiae expression system and Pichia pastoris expression system. In the last two decades, Pichia pastoris is intensive concerned, as its expression product will be directed into the medium, after recombinant proteins was set under the control of AOX1 promoters which is available to drive high-level expression, and fused to the α-factor or a native secretion signal for efficient secretion. To gain high purity of product like vaccine, many modern sophisticated hyphenated tools are developed，such as GC-MS, LC-MS, CE-MS, SFC-MS, LC-NMR, CE-NMR, LC-FTIR, etc. Considering the contaminant proteins are mainly from the host cell,if we eliminate the contaminant proteins by knockout or modificate, we will easily develop a simplified and cost-saving purification strategy. In this paper, Pichia pastoris GS115 was chosen as research subjects, and contaimiant proteins binding to Ni-IDA columns were identifie