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。目前已有的毕赤酵母表面展示系统主要是利用来自酿酒酵母Flol蛋白的N端絮凝结构域和GPI系统的C端为锚定蛋白将外源蛋白展示于菌体表面,融合蛋白分别非共价和共价连接于毕赤酵母细胞壁上。
为了优化毕赤酵母展示系统,本实验室成功采用三种锚定系统将CALB (Candida Antarctica lipase B)展示在毕赤酵母细胞(Pichia pastoris)表面,本论文在此基础上,进行了以下研究：
1.不同毕赤酵母表面展示南极假丝酵母脂肪酶B的比较,western blot证明融合蛋白KFS-CALB/GS115可被热SDS从重组酵母细胞壁提取,并大量分泌至培养基,KNS-CALB/GS115和Sed1-CALB/GS115可用葡聚糖酶提取,分泌能力较弱。无论何种锚定系统,其目的蛋白的实际分子量均远远大于理论分子量,这可能与融合蛋白的糖基化有关。流式细胞仪检测发现,三种锚定蛋白的重组毕赤酵母菌株的荧光强度明显不同,KFS强度最大,Sed1最小,同水解活力分析相同。以上结果说明相同宿主,相同外源蛋白,不同锚定系统的锚定效率有明显差异。对发酵液的水解活力和蛋白含量分析发现,KFS的分泌能力最强,可能同其的非共价结合方式有关。另外,KFS发酵液中的小分子融合蛋白片段表明目的蛋白的分泌可能同发酵过程中的蛋白酶有关。
2.絮凝素毕赤酵母展示系统展示机理的初探。通过单因子实验,确定了KFS-CALB/GS115展示系统最佳的与蛋白酶活性相关的营养和环境条件：诱导温度为28℃,蛋白胨的最佳添加量为2%。Gly、Ala和Pro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%。但随着展示活力的提高,分泌水平也随之提高。对KFS-CALB/GS115毕赤酵母和GS115的发酵液蛋白进行双向电泳发现,在大分子区出现融合蛋白点,经MALTI-TOF-TOF-MS测定为重组的融合蛋白,其分子量为216kD,等电点为3.97。
本研究对毕赤酵母展示系统的展示能力进行进一步分析,发现以N端絮凝素端结合的KFS重组酵母展示系统无论从其菌体表面还是发酵液均具有最大脂肪酶表达能力,具有较高可塑性。改变毕赤酵母的培养条件对融合蛋白的表达有极大影响。
The yeast pichia pastoris is a useful eucaryon expressing system which has developed in these yeasrs. This system has several good quality, for example, high amouts expression, clutivating low, product can secrete outside,proper glycosylation. At present, P.pastoris has been studied for protein-surface display based on the anchor system of the flocculation functional domain of Follp(FS) from S.cerevisiae and the C domain of GPI anchor system which were noncovalently and covalently linked to the cell wall respectively.
On the basis of previously work, laboratory constructed the gene of CALB which was displayed on the pichia pastoris using three different anchor systems.On this basis,the article is divided into two parts.
1.Comparing the display and secreion ability of foreign protein in different pichia pastoris display system.Western blot confirmed that the fusion proteins KFS-CALB/GS115 were released from cell walls after mild alkaline treatment,and can excrete into fermentation supernatant abundantly, conversely,the proteins KNS-CALB/GS115 and Sedl-CALB/GS115 can only extracted using glucanase with less secretory capacity. No matter which anchor system, the practical molecular weight of interest protein is more larger than its in theory which maybe is associated with glycosylation of fusion protein. The fluorescence intensity of the whole yeast was measured by flow cytometry, in which we found that three yeast strain with different anchor system had different fluorescence intensity that KFS had maximum and Sedl was smallest. The result indicate that the anchor efficiency was variance obviously with different anchor system even in the same host and the same foreign protein. To analyse the hydrolyse activity and protein concentration of fermentation supernatant we found that the high secretion capability of KFS was related to its noncovalent bonding possibility. Moreover, the micromolecule band in superanant of KFS showed that the excreted protein was associated with protease probablely.
2.The analyse of CALB in pichia display system with KFS anchor system. By the experiment of mono-factor,the main results as follows:under 28℃the expression of the protein is the highest. and 2% peptone was added to culture medium is the best, at the same time, Additions of Gly, Ala, Pro into the medium,0.1% concentrationhas the optimization expression. Neverthelessly, the serection level was increasing along with the raising of display ability. The 2DGE of the supernatant of KFS pichia pastoris and GS115 showed that the protein were concentrated in the acidity terminal and we can see the fusion protein in the macromolecule place.
The study analyze the display capability of pichia pastoris display system further, in which we found the KFS recombinant yeast that incorporated with flocculation anchor system had the biggest expression ability of the fusion lipase,either in the surface of cell or the fermentation supernate,and had the highest plasticity.lt had a big influence by the expression of fusion protein to change cultivation conditon.
引文
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