毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

展示南极假丝酵母脂肪酶B的毕赤酵母全细胞催化合成短链芳香酯

展示酶的酵母细胞作为全细胞催化剂,既具有固定化酶的优点,又有制备简单、成本较低的特点。本研究将细胞表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的重组毕赤酵母用于非水相中催化合成短链芳香酯,通过滴定和气相色谱的方法测定底物酸的转化率,从底物的碳链长度、醇的结构、酵母冻干粉的添加量、底物浓度及底物的酸醇摩尔比等方面考察了展示CALB的毕赤酵母全细胞催化合成短链芳香酯的特性。研究结果表明:该全细胞催化剂可催化C10以下的酸和醇直接酯化合成多种短链芳香酯,酸的转化率达到90%以上;其中己酸和乙醇为酶的最适底物;酵母冻干粉的添加量20g/L(306.0U/g-drycell)、己酸浓度0.8mol/L、酸醇摩尔比1:1.1是合成己酸乙酯的最佳条件。在此条件下反应1.5h,己酸的转化率达到97.3%。在现有的关于脂肪酶非水相催化合成短链芳香酯的报道中,该全细胞催化剂显示出较好的底物耐受性以及较高的催化反应速率。因此,展示CALB的毕赤酵母全细胞催化剂在合成短链芳香酯方面具有较大的商业化应用潜能。     

酵母表面展示酶技术

酵母表面工程是利用载体蛋白将外源蛋白以活性形式锚定于酵母细胞外表面,免去了外源蛋白的纯化和固定,并且对其有稳定作用。本文综述了酵母表面展示技术的原理、步骤、优点以及目前常见的酵母表面展示酶,如淀粉水解酶、纤维素水解酶、与木糖利用相关的酶、脂肪酶、有机磷水解酶的构建及应用。     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

利用Sed1p锚定蛋白在毕赤酵母表面展示米黑根毛霉脂肪酶及其应用

通过PCR扩增米黑根毛霉脂肪酶基因,在米黑根毛霉脂肪酶N端加入Flag标签。将米黑根毛霉脂肪酶基因与酿酒酵母细胞壁蛋白Sed1p基因的N端融合构建质粒pPIC9K-Flag-RML-Sed1,转化毕赤酵母GS115获得重组菌GS115/pPIC9K-Flag-RML-Sed1。重组菌经过甲醇诱导表达后,显微镜免疫荧光分析与流式细胞仪检测结果均证实米黑根毛霉脂肪酶已经成功展示在毕赤酵母上。该重组菌水解活力达到169.6U/g(Dry cell weight),在非水相中催化脂肪酸甲酯的合成,72h后脂肪酸甲酯的产率达82.36%。     

南极假丝酵母脂肪酶A的表面展示及酶学性质研究

采用a凝集素作为载体蛋白,首次将南极假丝酵母脂肪酶A展示在酿酒酵母细胞表面,通过MD平板筛选获得表面展示型的CALA酵母工程菌株。免疫荧光检测显示CALA被成功展示在酵母细胞壁表面,重组子经诱导后能在三丁酸甘油酯板上形成透明圈,说明展示的CALA具有活性。重组酵母在液体培养基培养72 h,活性达到最高,为80.4 U/g干细胞。酿酒酵母展示的CALA最适温度及pH值为70°C和pH 8.0。经50°C保温2 h,仍含有60%水解酶活力。展示的CALA在pH 7.0和pH 8.0溶液中比较稳定。经DMSO处理2 h,展示的CALA仍保持70%的活性。以上结果表明酵母展示的CALA可作为一种有潜质商业用途的全细胞催化剂。     

酵母表面展示技术在蛋白质工程中的应用

酵母细胞表面展示技术目前已成为蛋白质工程研究的重要工具,利用此技术可以鉴定蛋白间的相互作用、提高蛋白的亲和力和特异性、增加蛋白的稳定性和表达水平、绘制功能性抗原位图、固定化表达具有生物活性的蛋白和酶等,此项技术的运用代表着蛋白质工程研究中的最新进展.     

白地霉脂肪酶在毕赤酵母表面展示

将白地霉脂肪酶基因N端与酿酒酵母FLO絮凝结构域序列融合,构建成脂肪酶毕赤酵母表面展示载体并转化毕赤酵母GS115。免疫荧光检测证实脂肪酶已展示于毕赤酵母细胞表面。甲醇诱导96 h后展示酶活性达到81 U/g干细胞,酶的热稳定性较游离酶有较大提高,50℃孵育4 h后酶活仍保持初始酶活70%以上。     

解脂耶氏酵母脂肪酶Lip2的表面展示及其酶学性质

表面展示酶作为全细胞催化剂具备诸如能提高酶的稳定性、省去纯化过程、节约成本等优点。脂肪酶是应用最为广泛的工业酶之一。本研究利用酿酒酵母细胞壁蛋白Cwp2作为锚定蛋白,将解脂耶氏酵母脂肪酶Lip2展示在酿酒酵母细胞表面,以制备脂肪酶全细胞催化剂。Lip2被融合到Cwp2的N端,Cwp2通过其C端的GPI锚定信号共价结合到细胞壁上。表面展示的Lip2可以水解三丁酸甘油酯及对硝基苯酚辛酸酯(pNPC),其pNPC水解酶活达到4.6U/g干细胞。作为全细胞催化剂,表面展示的Lip2具备良好的催化特征,其最适温度为40°C,最适pH为8.0,同时还具备良好的有机溶剂稳定性。     

Yeast surface display of full-length human microtubule-associated protein tau

Microtubule-associated protein tau is an intrinsically disordered, highly soluble protein found primarily in neurons. Under normal conditions, tau regulates the stability of axonal microtubules and intracellular vesicle transport. However, in patients of neurodegeneration such as Alzheimer's disease (AD), tau forms neurofibrillary deposits, which correlates well with the disease progression. Identifying molecular signatures in tau, such as posttranslational modification, truncation, and conformational change has great potential to detect earliest signs of neurodegeneration and develop therapeutic strategies. Here, we show that full-length human tau, including the longest isoform found in the adult brain, can be robustly displayed on the surface of yeast Saccharomyces cerevisiae. Yeast-displayed tau binds to anti-tau antibodies that cover epitopes ranging from the N-terminus to the 4R repeat region. Unlike tau expressed in the yeast cytosol, surface-displayed tau was not phosphorylated at sites found in AD patients (probed by antibodies AT8, AT270, AT180, and PHF-1). However, yeast-displayed tau showed clear binding to paired helical filament (PHF) tau conformation-specific antibodies Alz-50, MC-1, and Tau-2. Although the tau possessed a conformation found in PHFs, oligomerization or aggregation into larger filaments was undetected. Taken together, yeast-displayed tau enables robust measurement of protein interactions and is of particular interest for characterizing conformational change.     

Establishment of cell surface engineering and its development

Cell surface display of proteins/peptides has been established based on mechanisms of localizing proteins to the cell surface. In contrast to conventional intracellular and extracellular (secretion) expression systems, this method, generally called an arming technology, is particularly effective when using yeasts as a host, because the control of protein folding that is often required for the preparation of proteins can be natural. This technology can be employed for basic and applied research purposes. In this review, I describe various strategies for the construction of engineered yeasts and provide an outline of the diverse applications of this technology to industrial processes such as the production of biofuels and chemicals, as well as bioremediation and health-related processes. Furthermore, this technology is suitable for novel protein engineering and directed evolution through high-throughput screening, because proteins/peptides displayed on the cell surface can be directly analyzed using intact cells without concentration and purification. Functional proteins/peptides with improved or novel functions can be created using this beneficial, powerful, and promising technique.     

酵母表面展示技术应用的最新进展

酵母表面展示(yeast surface display, YSD)技术是一种将外源靶蛋白基因序列与特定的载体基因序列融合后导入酵母细胞,利用酵母细胞内蛋白转运机制将靶蛋白表达并定位于酵母细胞表面的技术,最常用的是α-凝集素表达系统。酵母细胞具有真核细胞翻译后修饰机制,能够帮助目的蛋白正确折叠,可以用来展示各种真核蛋白,包括抗体、受体、酶和抗原肽等。酵母表面展示技术已成为生物技术和生物医学领域的强大蛋白质工程工具,结合流式细胞分选可用于改善蛋白质性质,包括亲和力、特异性、酶功能和稳定性等。本文从文库构建与筛选、抗体工程、蛋白质工程、酶工程和疫苗开发等方面对酵母表面展示技术应用最新进展进行了综述。     

酵母表面展示分选酶底物用于分选酶活性检测

摘要：【目的】以EGFP标签检测分选酶底物QALPETGEE在毕赤酵母表面的表达,然后将酵母表面展示的底物与分选酶相互作用以检测分选酶活性。【方法】以pcDNA-myc-his-EGFP为模板,通过PCR技术将QALPETGEE-linker-EGFP基因连接到穿梭载体pKFS上,构建QALPETGEE-linker-EGFP酵母表面展示载体后转化至毕赤酵母（Pichia pastoris）GS115中。重组菌经培养,利用荧光显微镜检测重组酵母的荧光强度,然后通过荧光分光光度计检测分选酶与底物相互作用后产     

Post-translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display

Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expected from expression levels and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing.     

利用乳酸乳球菌AcmA表面展示β-1,3-1,4-葡聚糖酶

采用PCR扩增乳酸乳球(Lactococcus lactis)MBl91菌株的全长肽聚糖水解酶基因acmA,通过C-末端融合构建了与绿色荧光基因gfp的融合基因acmA-gfp,再连接于表达载体pMG36k上后得到可组成型表达AcmA-GFP融合蛋白的重组质粒pMB137,然后将该质粒电转化导入到乳酸乳球菌AS1.2829中获得重组菌MB137.经SDS-PAGE检测.重组菌MB137可表达预期的分子量约74 kD的蛋白质.Western blotting、细胞分级分离组分的荧光活性测定和特异GFP 二抗标记的流式细胞仪检测证实GFP被成功锚定在重组茵细胞表面,被锚定蛋白约占总表达融合蛋白的35%.进一步通过从枯草芽胞杆菌BF7658基因组中扩增去信号肽序列的β-1,3-1,4葡聚糖酶基因gls,来取代pMB137中的gfp,得到携带融合基因acmA-gls的重组质粒pMB138,经导入到乳酸乳球茵AS1.2829后得到重组菌MB138,其全细胞β-1,3-1,4-葡聚糖水解酶的活性约为12 U/mL茵液,明显高于对照茵株.     

Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions

Yeast surface display, a well‐established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N‐ or C‐terminus of the yeast Aga2p mating protein. Historically, yeast‐displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N‐terminus and one to the C‐terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody‐staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein‐protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co‐expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.