摘要
毕赤酵母是近十年来发展起来的一种真核表达体系,具有表达量高、培养成本低、产物可以分泌到胞外、糖基化程度适中等优点。目前,国内外已有500多种外源蛋白在毕赤酵母中得到表达。但是外源蛋白的降解是毕赤酵母表达体系应用过程中的共性问题,严重影响外源蛋白的积累。
本研究室成功构建了IFNβ与人血清白蛋白融合蛋白(Human IFNβ-HSA fusion protein, hIFNβ-HSA)基因并在毕赤酵母(Pichia pastoris KM71)中表达,经检测融合蛋白分子量约为90 kDa,具有HSA的抗原性,用细胞病变抑制法测定摇瓶发酵液上清中融合蛋白的干扰素活性约为640 IU/mL,并通过对5 L发酵罐发酵过程参数及其过程控制对基因工程菌毕赤酵母表达hIFNβ-HSA影响的研究确定一系列的发酵参数。本论文是在此工作基础上,选取的主要结果如下:
1.通过单因子实验,确定了最佳的与蛋白酶活性相关的营养和环境条件:pH在5.0-6.0,蛋白胨的最佳添加量为6%。Glu、Ala和Arg三种氨基酸的添加会促进融合蛋白的表达。其最佳作用浓度为0.1%。最后确定了EDTA为促进融合蛋白表达的最适蛋白酶抑制剂,其最佳作用浓度为10 mmol/L。根据Box-Benhnken的中心组合试验设计原理,采用三因素三水平的响应面分析方法,考察了pH、Glu浓度和EDTA浓度对融合蛋白表达的影响,结果表明当pH 5.5、添加Glu浓度为0.18%、EDTA浓度为12 mmol/L时,融合蛋白的表达量最高,为24.87 mg/L,相对于对照组提高了61%。
2.通过SDS-PAGE确定融合蛋白hIFNβ-HSA在诱导表达过程中确实发生了降解,降解的主要条带出现在65 kDa;通过Western-Blotting杂交实验,证实了65 kDa的蛋白条带与90 kDa的目的条带在杂交实验中具有相同的蛋白免疫原性,从而确定这条蛋白带为90 kDa目的条带的降解带。通过HPLC证实发酵液中蛋白酶对融合蛋白的降解主要发生在HSA区域。以明胶为底物的SDS-PAGE活性电泳结果表明,诱导表达阶段的发酵上清液和细胞内存在几种蛋白酶。以HSA为底物的SDS-PAGE活性电泳,确定了在发酵液中确实存在一种蛋白酶对融合蛋白进行水解作用。在此基础上,对与标准蛋白分子量Mark进行比较,确定该蛋白酶为蛋白酶B。
The yeast Pichia pastoris is a useful eucaryon expressing system which has developed in 10 years. This system has several good quality , for example, high amouts expression, clutivating low, product can secrete outside, proper glycosylation. At present, already have had more than 500 kind external protein expressed in Pichia pastoris. Proteolytic degradation has been a perpetual problem when yeasts are employed to express recombinant proteins. Alos this problem influent the accumulation of protein.
On the basis of previously work, laboratory constructed the gene of Human IFNβ-HSA fusion protein, hIFNβ-HSA in Pichia pastoris KM71 by technique of gene engineering, which express the 90 kDa protein. And tthrough he effects of fermentative process parameter on cell growth and hIFNβ-HSA expression in 5 L fermenter were studied. On this basis , this research is about the problem of the Proteolytic degradation during the express process, the main results as follows:
1. By the experiment of mono-factor, the main results as follows: under the pH5.0~pH6.0 the exprrssion of the protein is the highest, and 6% peptone was added to culture medium is the best, at the same time, Additions of Glu, Ala, Arg into the medium, 0.1% concentration has the optimization expression. According of the principle of Box-Benhnken, with the method of response surface of the three factors and three levels, the best addition program is pH5.5、Glu 0.18%、EDTA 12 mmol/L in the medium, obtained the amount of protein IFNβ-HSA is 24.87 mg/L, comparing with the blank referenc group, increasing 61%
2.. The analysis of SDS-PAGE and Western-blot showed that 90kDa fusion protein and 65kDa degradation band appeared in the fermentation. By the gelatin-SDS-PAGE, three prteases inside and five proteases outside cell, and which degrdated the pritein. With the method of HPLC indicated that the degradation appeared in the HSA region of recombinant human IFNβ-HSA fusion protein. Applied by SDS-PAGE activity electrophoresis the protease which caused the degradation of fusion protein IFNβ-HSA was proteinase B
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