重组双碱基内肽酶在毕赤酵母中的表达制备及酶活鉴定

发布时间：2017-12-27 23:20

本文关键词：重组双碱基内肽酶在毕赤酵母中的表达制备及酶活鉴定　出处：《重庆理工大学》2016年硕士论文　论文类型：学位论文

【摘要】：Kex2即双碱基内肽酶,也称为Kexin、Paired-basic endopeptidase、Prohormone-processing endoprotease等,这些名称从不同的角度反应了Kex2的酶学性质。Kex2基因来自酿酒酵母Saccharomyces cerevisia,属于枯草杆菌蛋白酶家族,是一个钙离子依赖型的丝氨酸蛋白水解酶。Kex2能特异性识别蛋白质或多肽中的双碱性氨基酸残基对(Lys-Arg、Arg-Arg)及Pro-Arg,从第二个氨基酸残基羧基端(即R-)切断肽键,发挥作用。Kex2作为前体加工酶的原型,对其的研究大大促进了其他各种真核生物前体加工酶的研究进步。另外,人们还发现Kex2能在体内和体外环境下完成对于一些激素原前体、蛋白前体的酶切加工。所以,由于其酶切位点的特异性,近几年Kex2在生物制药领域逐渐显示地位。要想深入进行上述研究,首先必须得到大量Kex2酶,才能进行体内和体外各项研究。目前对于Kex2的结构及酶活性等研究已经较为全面,而对于Kex2的重组构建及制备的研究还较少。虽然已有相关文献报道,但是能够筛选到高表达量的重组工程菌,用于发酵并完成后续纯化过程得到Kex2纯品的研究相对较少,本文致力于完成这一研究目标和内容。研究目的:本项目中创新地设计重组双碱基内肽酶Kex2蛋白序列和应用于毕赤酵母系统表达的核苷酸序列,构建能够正确表达Kex2酶的毕赤酵母重组表达菌从而提高Kex2产量;摸索重组表达菌上罐发酵工艺,为工业化生产打下基础;建立稳定、重复性好、回收率高的纯化工艺,以得到Kex2纯品;建立Kex2浓度测定方法、活性测定方法,并将其用于蛋白类底物的酶切实验,以验证其活性。研究方法:1重组Kex2 c DNA序列的设计参阅文献,选择Kex2蛋白序列2-660残基,并在N端设计His标签LEKRSARGSHHHHHH以利于下游纯化,;并根据P.Pastoris密码子使用偏好性,设计了Kex2的c DNA,设计终止密码子TGA和TAA,设计上下游酶切位点Xho I(CTCGAG)和Not I(GCGGCCGC)限制性酶切位点。2重组表达菌pPICZαA-Kex2/X-33的构建全基因合成cDNA序列,得到含有目的序列的甘油菌。抽提质粒,Xho I和Not I双酶切后连入载体p PICZαA,转化Top10F’感受态细胞,筛选后得到重组质粒p PICZαA-Kex2。用Sac I线性化重组质粒,电转化进入X-33感受态细胞,挑取阳性克隆进行甲醇诱导表达,利用抗性平板等方法筛选高表达菌株,并对其进行鉴定,确定为发酵用表达菌。3发酵工艺建立初期用摇瓶发酵的方式来筛选重组表达菌最适温度、p H、溶氧等条件,随后上罐发酵,摸索并建立发酵工艺,确定发酵培养基、诱导时间、诱导方式等等。4发酵上清的纯化工艺建立根据设计蛋白序列的创新性,优选试用Ni离子金属鳌合柱进行初纯化。后选择离子柱进行纯化,去掉多余的杂质。5自制Kex2样品酶切活性鉴定针对于Kex2酶切特异性选择特异性测活底物、短肽、蛋白类三种不同底物,建立各自酶切方法,分别验证自制Kex2酶切活性和特异性。研究结果:本研究成功构建了pPICZαA-Kex2/X-33重组表达菌,完成了Kex2在毕赤酵母系统的重组表达;对筛选出的表达量最高的菌株完成了10L上罐发酵,并创新地采用Ni离子金属鳌合柱初纯化、脱盐柱处理后阴离子柱再纯化,得到了电泳纯度95%以上的重组Kex2样品;随后对样品进行了浓度和纯度检测,最重要的是对酶切效率和特异性进行了三种底物水平的检测,证明自制的Kex2样品在以特异性底物Boc-QRR-p NA、短肽(含有KR识别位点)、蛋白质水平(甘精胰岛素前体,含有KR和RR识别位点)都可以发挥其酶切作用,且不会发生错切现象,这对Kex2今后的应用及Kex2的工业化生产都有极大的意义。
[Abstract]:Kex2 double alkali medium peptide enzyme, also known as Kexin, Paired-basic endopeptidase, Prohormone-processing endoprotease, the name of the reaction of Kex2 properties from different angles. The Kex2 gene is derived from Saccharomyces cerevisia of Saccharomyces cerevisiae, belonging to the family of Bacillus subtilis protease, and is a calcium dependent serine protein hydrolase. Kex2 can identify specific protein or polypeptide in two basic amino acid residues of (Lys-Arg, Arg-Arg) and Pro-Arg, from the second amino acid C-terminal (R-) cut the peptide bond, play a role. As a precursor of precursor processing enzymes, the research of Kex2 has greatly promoted the progress of various other eukaryotic precursor processing enzymes. In addition, it is also found that Kex2 can perform enzyme cutting for some hormone precursors and protein precursors in the body and in vitro. Therefore, due to the specificity of the enzyme site, Kex2 has gradually shown its status in the field of biopharmaceutical in recent years. In order to carry out the above research, we must first obtain a large number of Kex2 enzymes, in order to carry out in vivo and in vitro studies. At present, the research on the structure and enzyme activity of Kex2 has been more comprehensive, but there are few studies on the construction and preparation of Kex2. Although there are related reports, it is possible to screen high expression recombinant engineered bacteria, which can be used for fermentation and subsequent purification process to get Kex2 pure products. Objective: To study the creative designing nucleotide sequence of recombinant double alkali medium peptidase Kex2 and protein sequences used in Pichia pastoris expression system in the project construction, to the correct expression of enzyme Kex2 in Pichia pastoris recombinant bacteria to improve the yield of Kex2; recombinant expression fermentation process can find bacteria, lay the foundation for industrial production.; to establish a stable, good repeatability and high recovery rate and purification process, in order to obtain pure Kex2; establishment of Kex2 concentration determination method, activity determination method, and used for protein substrate enzyme digestion experiments to verify its activity. Research methods: 1 recombinant Kex2 C DNA sequence of the literature, select the Kex2 protein sequence of 2-660 residues, and in the end N His label design LEKRSARGSHHHHHH to the downstream purification; and according to the P.Pastoris codon preference, the design of Kex2 C DNA, the design of the termination codon TGA and TAA, the design of the downstream enzyme Xho I restriction sites (CTCGAG) and Not I (GCGGCCGC) restriction sites. 2 the recombinant expression bacteria pPICZ alpha A-Kex2/X-33 was constructed to synthesize the whole gene cDNA sequence, and the glycerol containing the target sequence was obtained. The plasmid, Xho I and Not I were cut into the carrier P PICZ alpha A, and transformed Top10F 'receptive cells, and the recombinant plasmid P PICZ alpha A-Kex2 was obtained after screening. Of the recombinant plasmid with Sac I linear, was transformed into X-33 competent cells. The positive clones were induced by methanol, the use of resistant plate method for screening high expression strains, and to identify its expression as determined by bacteria fermentation. 3, in the early stage of fermentation, the best temperature, P H, dissolved oxygen and other conditions were screened by shake flask fermentation. Then the fermentation process was carried out on the top tank, and the fermentation process was established, and the fermentation medium, induction time, induction mode and so on were determined. 4 the purification process of the fermentation supernatant was established on the basis of the innovation of the designed protein sequence, and the Ni ion metal chelating column was first purified. Then the ion column was selected to be purified to remove the excess impurities. 5 homemade Kex2 sample digestion activity identification for Kex2 enzyme specific specificity assay Chet substrates, peptides, proteins of three different substrates, establish their own digestion method were validated homemade Kex2 enzyme activity and specificity. Results: This study successfully constructed pPICZ alpha A-Kex2/X-33 recombinant expression bacteria, completed the expression of Kex2 in Pichia pastoris recombinant; on expression of the highest strains completed 10L fermentation, and innovative use of Ni metal chelate column initial purification, desalination column after column purification and anion that was obtained more than 95% purity of recombinant Kex2 samples; the samples were subsequently subjected to concentration and purity detection, the most important is the efficiency and specificity of enzyme cutting were detected three kinds of substrate level, proved that the homemade Kex2 samples in a specific substrate Boc-QRR-p, NA peptide (containing KR recognition sites), the protein level (insulin glargine precursor containing KR and RR recognition sites) can play the role of digestion, and can not have the wrong cut phenomenon, are of great significance for industrial production and application of the Kex2 Kex2 in the future.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R915

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