【作者】 王庆杰；

【导师】 王鹏；

【作者基本信息】 山东大学 ， 微生物学， 2008， 博士

【摘要】 随着基因工程的发展,微生物表面展示系统已发展出噬菌体展示系统、细菌表面展示系统、酵母表面展示系统等,广泛应用在生物学研究及工业生产的许多领域,例如研究蛋白质-蛋白质之间的相互识别与相互作用、由多肽构象或蛋白质突变库中获取特定功能蛋白、生产抗体、生产口服疫苗、开发全细胞催化剂、开发生物传感器、开发细胞吸附剂等等,成为竞相研究的热点。但已发展的微生物表面展示系统都存在自身明显的缺陷:噬菌体展示系统展示的多肽或蛋白分子量不能太大,否则会影响噬菌体的装配与感染;革兰氏阴性菌表面展示系统表达的重组蛋白易以包涵体形式存在,展示的蛋白不仅要通过质膜层,还要在外膜上正确的整合,这可能对展示蛋白的结构和活性产生影响;革兰氏阳性菌细胞壁厚,转化效率较低,这样会影响大容量肽库的构建;两种细菌表面展示系统均缺乏翻译后修饰功能,容易使展示的真核蛋白失活;酿酒酵母中虽存在蛋白质翻译后修饰,但糖基化方式与哺乳动物不同,它对外源蛋白N-糖基化修饰形式是高甘露糖型,糖链平均为50-150甘露糖残基,并且核心寡糖末端是α-1,3-甘露糖,从而使生产的蛋白在人体中有比较强的免疫原性,过度糖基化还可能使表达的酶等功能性蛋白失活。毕赤酵母(Pichia pastoris)表达系统是近年来发展迅速、应用广泛的一种真核表达系统。其具有易于培养、繁殖快、具有乙醇氧化酶基因诱导型强启动子,可调控外源基因的表达、可对外源蛋白进行更接近高等生物的加工折叠和翻译后修饰、便于基因工程操作和高密度发酵等的特性,成为微生物表面展示系统极有应用前景的宿主菌。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Flo1蛋白的N端絮凝结构域为锚定蛋白将外源蛋白展示于菌体表面,融合蛋白非共价连接于毕赤酵母细胞壁上。展示的外源蛋白通过N端与锚定蛋白融合,C端蛋白游离,这样的系统适合脂酶等活性位点在C端的功能蛋白。但对活性位点在N端的功能蛋白表达不利,限制了更广泛的应用。为丰富毕赤酵母展示系统,展示不同需要的外源蛋白,本文做了以下3部分工作:第一部分:Pir1型毕赤酵母表面展示系统的构建及表达研究Pir(Proteins with internal repeats)系列蛋白是酿酒酵母中共价连接细胞壁的甘露糖蛋白,C端无保守的GPI锚定序列,直接与细胞壁中β-1,3-葡聚糖相连。成熟肽蛋白N端有数目不等的重复序列,可被温和碱(30 mM NaOH,12h,4℃)从细胞壁抽提。外源蛋白可通过与Pir蛋白N端融合、C端融合或插入融合展示在菌体表面。目前Pir系列蛋白在酿酒酵母表面已成功展示了多种外源蛋白。本研究克隆了来自酿酒酵母EBY100的Pir1蛋白成熟肽基因(Pir1-a),连接入毕赤酵母分泌表达载体pPIC9K中,构建了新型毕赤酵母表面展示的通用载体pPIC9K-Pir1-a。利用α-factor信号肽将蛋白引导分泌至细胞外。通过免疫荧光分析以C端与Pir1-a蛋白融合而展示于毕赤酵母表面的增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP),证明新的毕赤酵母表面展示系统的构建是成功的。但激光荧光共聚焦显微镜(confocal laser scanning microscopy,CLSM)观察到EGFP-Pir1-a融合蛋白的展示是不规则的,分析这种不规则的展示可能与Pir1蛋白自身性质相关。进一步以Pir1成熟肽N端重复序列(Pir1-b蛋白)为锚定蛋白,构建新的毕赤酵母表面展示载体pPIC9K-Pir1-b,表面展示的模式蛋白仍采用EGFP。CLSM观察到新的融合蛋白EGFP-Pir1-b是规则的展示在重组毕赤酵母表面。Western blot证明了融合蛋白EGFP-Pir1-a与EGFP-Pir1-b可被温和碱从重组毕赤酵母细胞壁抽提,并且很少分泌到培养基中。荧光分光光度计及流式细胞仪检测发现,分别以Pir1-α和Pir1-b为锚定蛋白时,相同诱导条件下,表面展示EGFP的重组毕赤酵母菌株的荧光强度是相当的。以上结果说明酿酒酵母的Pir1蛋白可作为毕赤酵母的锚定蛋白,将外源蛋白展示于毕赤酵母表面,融合蛋白没有改变Pir1蛋白自身的锚定性质:同时说明了Pir1成熟肽蛋白中,其N端重复序列对其锚定在细胞壁起主要作用,C端序列对其在表面的不规则展示起主要作用。第二部分:α凝集素型毕赤酵母表面展示系统的构建及毕赤酵母表达重组蛋白纯化新方法的探讨α凝集素是是酿酒酵母中共价连接细胞壁的GPI(Glycosyl phosphatidylinositol)型甘露糖蛋白的一种,C端有GPI锚定序列,并通过β-1,6-葡聚糖连接至β-1,3-葡聚糖,可被β-1,3-或β-1,6-葡聚糖酶作用。外源蛋白可通过C端与α凝集素C末端融合表达而锚定在细胞表面。目前以α凝集素C末端为锚定蛋白在酿酒酵母表面已成功展示了多种外源蛋白。本研究克隆了来自酿酒酵母EBY100的α凝集素的C末端基因,连接入毕赤酵母分泌表达载体pPIC9K中,构建了新型毕赤酵母表面展示通用载体pPIC9K-AGα1。利用α-factor信号肽将外源蛋白引导分泌至细胞外,同时在锚定蛋白序列前增加了MluI、ApaI限制性内切酶位点及Xpress抗原序列,提供了肠激酶作用位点。通过免疫荧光分析以C端与α凝集素的C末端蛋白融合而展示于毕赤酵母表面的EGFP,证明新的毕赤酵母表面展示系统的构建是成功的。通过荧光显微镜观察及Western blot分析证明了肠激酶可将展示在重组毕赤酵母表面的EGFP与菌体分离,从而探讨了纯化毕赤酵母表达重组蛋白的新方法:外源蛋白可利用毕赤酵母的体系进行需要的折叠、翻译后修饰并分泌到胞外,通过锚定蛋白展示于菌体表面,经肠激酶作用,可从菌体表面分离下来。第三部分:两种毕赤酵母表面展示系统展示外源蛋白能力的比较在成功地以来自酿酒酵母的Pir1蛋白及α凝集素C末端为锚定蛋白构建了毕赤酵母表面展示系统的基础上,以通过C端分别与两种锚定蛋白融合的EGFP为报告蛋白,通过研究诱导温度、诱导时间、诱导培养基初始pH、甲醇诱导浓度等摇瓶培养的发酵条件,探讨不同发酵条件分别对两种重组毕赤酵母菌株表面展示融合蛋白的影响。并在两者各自最适的发酵条件下对其展示外源蛋白的能力进行比较,结果发现α凝集素C末端为锚定蛋白时,其展示通过C端与其融合的外源蛋白的能力强于Pir1成熟肽蛋白。更多还原

【Abstract】 With the application of recombinant DNA technology,cell-surface display systems have been developed in various microorganisms,such as in phage,in bacteria and especially in Saccharomyces cerevisiae.Microbial cell-surface display has been widely used in biotechnological research and industrial applications,including: identifying protein-protein interactions,displaying polypeptide libraries as selection devices,mapping functional protein epitopes,immobilizing proteins and enzymes as whole cell biocatalysts,producing antibodies and live vaccines,producing bioadsorbents for the removal of harmful chemicals and heavy metals,et al.There are obvious disadvantages in the present microbial cell-surface display systems.In phase-display system,when larger-sized polypeptides were fused to the major coat protein of the phage,the hybrids were not readily incorporated into the phage particle;In Gram-negative display systems,both translocation through the cytoplasmic membrane and correct integration into the outer membrane may affect the structure and function of the proteins.In Gram-positive bacteria display systems,the lower frequency of transformation restricts the creation of large combinatorial protein libraries.Both the Gram-negative display systems and Gram-positive bacteria display systems cann’t fold and glycosylate the eukaryotic protein.In S.cerevisiae display system,secreted proteins are hyperglycosylated and have 50-150 mannoses per N-glycan,which may affect the activity of enzyme.In addition,S.cerevisiae core oligosaccarides have terminalα-1,3-glycan linkages which may make proteins hyper-antigenic and unsuitable for therapeutic use.The methylotrophic yeast,Pichia pastoris,is a single-cell eukaryote microorganism and has been successfully used to produce various recombinant heterologous proteins for both basic laboratory research and industrial manufacture. The advantages for P.pastoris to express heterologous proteins are following:it is easy to genetically manipulate and culture and can be grown to high cell densities;it can perform higher eukaryotic post-translational modifications,such as glycosylation and disulphide bond formation for the produce of soluble,correctly folded recombinant proteins;it has strong promoters which can drive the expression of interest protein. P.pastoris has been studied for protein-surface display based on the anchor system from S.cerevisiae.Tanino et al.immobilized the lipase from Rhizopus oryzae with a pro-sequence on the P.pastoris cell surface using the flocculation functional domain of Flolp(FS)from S.cerevisiae as an anchor.This system was noncovalently linked to the cell wall and suitable for the enzymes possessing activities at the C terminus.It is unsuitable for the enzymes that possess activities at the N terminus, which maybe limit the application of P.pastoris as a prospective cellular host for protein-surface display.This article is divided into three parts.PartⅠ:Construction of a novel Pichia pastoris cell-surface display system based on the cell wall protein Pir1Pir proteins are the covalently linked cell wall proteins in S.cerevisiae,which attach directly to theβ-1,3-glucan of the cell wall.Pir proteins do not contain a C-terminal GPI sequence and they are all processed by Kex2 protease.They can be released from intact cells by very mild alkaline treatment(30 mM NaOH,12 h,4℃). All Pir proteins have one or several units of an internal repetitive sequence at the N terminus.The target proteins can be fused to Pir proteins either in the middle or at the N- or C-terminus of Pir proteins.Pir proteins have been efficient anchors for the display of target proteins on the surface of S.cerevisiae.A novel system based on Pir1 from S.cerevisiae was developed for cell-surface display of heterologous proteins in P.pastoris with the alpha-factor secretion signal sequence.As a model protein,enhanced green fluorescence protein(EGFP)was fused to the N-terminal of the mature peptide of Pir1(Pir1-a).The expression of fusion protein EGFP-Pir1-a was irregular throughout the P.pastoris cell surface per detection by confocal laser scanning microscopy.A truncated sequence containing only the internal repetitive sequences of Pir1-a(Pir1-b)was used as a new anchor protein in further study.The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface.The fluorescence intensity of the whole yeast was measured by spectrofluorometer.Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment.The results indicate that a Pir1-based system can express proteins on the surface of P.pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall.The repetitive sequences of Pir1 are required for cell wall retention,and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P.pastoris.PartⅡ:Construction of a novel system for cell surface display of heterologous proteins on Pichia pastorisα-agglutinin belongs to another covalently linked cell wall proteins in S. cerevisiae,which have a GPI anchor at their C terminus and are attached toβ-1,3-glucan of the cell wall via a shortβ-1,6-glucan.GPI proteins can be released byβ-1,3- orβ-1,6-glucanase from cell wall.The C-terminal half ofα-agglutinin has been widely used to anchor heterologous proteins on the surface of S.cerevisiae.A versatile vector was developed for heterologous proteins display on the cell surface of P.pastoris using the C-terminal half ofα-agglutinin from S.cerevisiae as a membrane anchor under the control of the alcohol oxidase 1 promoter(pAOX1). Multiple cloning sites and the sequence encoding the Xpress epitope (-Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys-)were introduced into the vector for insertion of heterologous genes and selective cleavage of target proteins.EGFP was used as a model protein to check the function of this vector.The expression of EGFP on the P. pastoris surface was confirmed by confocal laser scanning microscopy.Fluorescence microscopy and western blot analysis confirmed that EGFP can be successfully cleaved from the cell surface by treating with enterokinase.This system can be applied to purify heterologous proteins expressed in P.pastoris.PartⅢ:Comparison of cell wall proteins of Pir1 and the C-terminal half ofα-agglutinin as anchors for heterologous protein cell-surface displayThe mature peptide of Pir1 and the C-terminal half ofα-agglutinin have been proved capable of immobilizing EGFP on the cell surface of P.pastoris.The fraction of the total amount of fusion protein localized to the cell wall varied depending on both the anchor domain used and the fermentation conditions.The expression of fusion protein under various fermentation conditions,e.g.,the induction temperature, initial pH and methanol concentration were studied.And the ability of the mature peptide of Pir1 and the C-terminal half ofα-agglutinin to anchor EGFP to the cell wall of P.pastoris was compared under each of the optimal induction condition.It was found the constructed P.pastoris strain could display more fusion protein when the C-terminal half ofα-agglutinin was used as an anchor.更多还原