本文關(guān)鍵詞：重組雙堿基內(nèi)肽酶在畢赤酵母中的表達(dá)制備及酶活鑒定　出處：《重慶理工大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 重組雙堿基內(nèi)肽酶 發(fā)酵 蛋白純化 酶活鑒定 酶切特異性

【摘要】：Kex2即雙堿基內(nèi)肽酶,也稱(chēng)為Kexin、Paired-basic endopeptidase、Prohormone-processing endoprotease等,這些名稱(chēng)從不同的角度反應(yīng)了Kex2的酶學(xué)性質(zhì)。Kex2基因來(lái)自釀酒酵母Saccharomyces cerevisia,屬于枯草桿菌蛋白酶家族,是一個(gè)鈣離子依賴(lài)型的絲氨酸蛋白水解酶。Kex2能特異性識(shí)別蛋白質(zhì)或多肽中的雙堿性氨基酸殘基對(duì)(Lys-Arg、Arg-Arg)及Pro-Arg,從第二個(gè)氨基酸殘基羧基端(即R-)切斷肽鍵,發(fā)揮作用。Kex2作為前體加工酶的原型,對(duì)其的研究大大促進(jìn)了其他各種真核生物前體加工酶的研究進(jìn)步。另外,人們還發(fā)現(xiàn)Kex2能在體內(nèi)和體外環(huán)境下完成對(duì)于一些激素原前體、蛋白前體的酶切加工。所以,由于其酶切位點(diǎn)的特異性,近幾年Kex2在生物制藥領(lǐng)域逐漸顯示地位。要想深入進(jìn)行上述研究,首先必須得到大量Kex2酶,才能進(jìn)行體內(nèi)和體外各項(xiàng)研究。目前對(duì)于Kex2的結(jié)構(gòu)及酶活性等研究已經(jīng)較為全面,而對(duì)于Kex2的重組構(gòu)建及制備的研究還較少。雖然已有相關(guān)文獻(xiàn)報(bào)道,但是能夠篩選到高表達(dá)量的重組工程菌,用于發(fā)酵并完成后續(xù)純化過(guò)程得到Kex2純品的研究相對(duì)較少,本文致力于完成這一研究目標(biāo)和內(nèi)容。研究目的:本項(xiàng)目中創(chuàng)新地設(shè)計(jì)重組雙堿基內(nèi)肽酶Kex2蛋白序列和應(yīng)用于畢赤酵母系統(tǒng)表達(dá)的核苷酸序列,構(gòu)建能夠正確表達(dá)Kex2酶的畢赤酵母重組表達(dá)菌從而提高Kex2產(chǎn)量;摸索重組表達(dá)菌上罐發(fā)酵工藝,為工業(yè)化生產(chǎn)打下基礎(chǔ);建立穩(wěn)定、重復(fù)性好、回收率高的純化工藝,以得到Kex2純品;建立Kex2濃度測(cè)定方法、活性測(cè)定方法,并將其用于蛋白類(lèi)底物的酶切實(shí)驗(yàn),以驗(yàn)證其活性。研究方法:1重組Kex2 c DNA序列的設(shè)計(jì)參閱文獻(xiàn),選擇Kex2蛋白序列2-660殘基,并在N端設(shè)計(jì)His標(biāo)簽LEKRSARGSHHHHHH以利于下游純化,;并根據(jù)P.Pastoris密碼子使用偏好性,設(shè)計(jì)了Kex2的c DNA,設(shè)計(jì)終止密碼子TGA和TAA,設(shè)計(jì)上下游酶切位點(diǎn)Xho I(CTCGAG)和Not I(GCGGCCGC)限制性酶切位點(diǎn)。2重組表達(dá)菌pPICZαA-Kex2/X-33的構(gòu)建全基因合成cDNA序列,得到含有目的序列的甘油菌。抽提質(zhì)粒,Xho I和Not I雙酶切后連入載體p PICZαA,轉(zhuǎn)化Top10F’感受態(tài)細(xì)胞,篩選后得到重組質(zhì)粒p PICZαA-Kex2。用Sac I線性化重組質(zhì)粒,電轉(zhuǎn)化進(jìn)入X-33感受態(tài)細(xì)胞,挑取陽(yáng)性克隆進(jìn)行甲醇誘導(dǎo)表達(dá),利用抗性平板等方法篩選高表達(dá)菌株,并對(duì)其進(jìn)行鑒定,確定為發(fā)酵用表達(dá)菌。3發(fā)酵工藝建立初期用搖瓶發(fā)酵的方式來(lái)篩選重組表達(dá)菌最適溫度、p H、溶氧等條件,隨后上罐發(fā)酵,摸索并建立發(fā)酵工藝,確定發(fā)酵培養(yǎng)基、誘導(dǎo)時(shí)間、誘導(dǎo)方式等等。4發(fā)酵上清的純化工藝建立根據(jù)設(shè)計(jì)蛋白序列的創(chuàng)新性,優(yōu)選試用Ni離子金屬鰲合柱進(jìn)行初純化。后選擇離子柱進(jìn)行純化,去掉多余的雜質(zhì)。5自制Kex2樣品酶切活性鑒定針對(duì)于Kex2酶切特異性選擇特異性測(cè)活底物、短肽、蛋白類(lèi)三種不同底物,建立各自酶切方法,分別驗(yàn)證自制Kex2酶切活性和特異性。研究結(jié)果:本研究成功構(gòu)建了pPICZαA-Kex2/X-33重組表達(dá)菌,完成了Kex2在畢赤酵母系統(tǒng)的重組表達(dá);對(duì)篩選出的表達(dá)量最高的菌株完成了10L上罐發(fā)酵,并創(chuàng)新地采用Ni離子金屬鰲合柱初純化、脫鹽柱處理后陰離子柱再純化,得到了電泳純度95%以上的重組Kex2樣品;隨后對(duì)樣品進(jìn)行了濃度和純度檢測(cè),最重要的是對(duì)酶切效率和特異性進(jìn)行了三種底物水平的檢測(cè),證明自制的Kex2樣品在以特異性底物Boc-QRR-p NA、短肽(含有KR識(shí)別位點(diǎn))、蛋白質(zhì)水平(甘精胰島素前體,含有KR和RR識(shí)別位點(diǎn))都可以發(fā)揮其酶切作用,且不會(huì)發(fā)生錯(cuò)切現(xiàn)象,這對(duì)Kex2今后的應(yīng)用及Kex2的工業(yè)化生產(chǎn)都有極大的意義。
[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.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R915

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