基于冰核蛋白的乳酸菌表面展示系统的构建

[目的]验证来源于丁香假单胞菌的冰核蛋白在乳酸乳球菌表面展示外源蛋白的可能性.[方法]以绿色荧光蛋白(Green Fluorescence Protein,GFP)基因gfp为报告基因,以冰核蛋白基因的N末端和NC端作为展示单元,构建乳酸菌表面展示载体pHZ101和pHZ102,并转化大肠杆菌(Escherichia coli JM109和乳酸乳球菌(Lactococcus lactis)MG1363.[结果]荧光显微镜观察显示重组大肠杆菌和乳酸乳球菌均能检测到绿色荧光.Western blot结果表明GFP蛋白在重组大肠杆菌和乳酸乳球菌中均得到表达,并且INPN-GFP蛋白多数滞留于乳酸乳球菌细胞质内,而INPNC-GFP蛋白则大部分定位于乳酸乳球菌的细胞膜上.[结论]以上结果表明丁香假单胞菌的冰核蛋白能引导外源蛋白定位于乳酸乳球菌的细胞膜上,为乳酸菌表面展示系统的构建提供了新的方向.     

利用冰核蛋白N-端结构域在大肠杆菌表面展示人存活蛋白

存活蛋白(survivin)是高度特异的广谱肿瘤相关抗原,利用冰核蛋白(ice nucleation protein,INP)表面展示系统研究在大肠杆菌细胞表面展示人存活蛋白的可行性。将人存活蛋白基因片段和报告基因Cherry融合到冰核蛋白N-端,构建表面展示载体p ET-INP-CHY-SUV并转化大肠杆菌BL21(DE3)。重组菌经IPTG诱导后可检测到红荧光,荧光强度在胰蛋白酶的作用下降低,完整细胞ELISA实验及免疫荧光实验可检测和观察到绿荧光,表明人存活蛋白在重组菌中得到表达,并且成功展示于细胞表面,为进一步研制新型的肿瘤DNA疫苗奠定基础。     

细菌运载蛋白及在表面展示技术中的应用进展

细菌细胞表面展示技术是一项新的蛋白质应用技术,其体系由运载蛋白、靶蛋白和宿主菌三者构成,一般可将其分为革兰阴性菌展示体系和革兰阳性菌展示体系两大类。目前已证实多种具有锚定活性的运载蛋白,并用于不同靶蛋白的细胞表面展示体系。该技术现已被应用于活体重组疫苗的开发、蛋白质文库构建与筛选、生物传感器、全细胞生物催化剂、全细胞生物吸附与降解等多个研发领域。     

细菌脂肪酶在大肠杆菌细胞表面的功能性展示

细胞表面展示技术已广泛应用于突变文库的高通量筛选,有力地促进了蛋白质工程的发展。以来自于铜绿假单胞菌的自转运蛋白Est A的羧基端结构域作为锚定区,构建脂肪酶LipA与EstA羧基端结构域的融合基因,并将融合基因插入到改造后的pACYC-Duet表达载体中,获得表面展示载体pBCCB-X1。将载体pBCCB-X1分别导入到大肠杆菌JK321和大肠杆菌UT5600菌株中,以IPTG诱导融合基因的表达。分别用三丁酸甘油酯定性检测和pNPO定量检测诱导表达后的全细胞脂肪酶的水解活性。试验结果表明,脂肪酶LipA在大肠杆菌JK321和大肠杆菌UT5600细胞表面均得到功能性展示,水解活性分别为(2.8±0.1)U/OD和(2.6±0.06)U/OD。脂肪酶LipA在大肠杆菌细胞表面的功能性展示,为后续高通量筛选LipA突变基因文库,奠定了坚实的基础。     

利用S-层蛋白在细胞表面展示α-淀粉酶和金属硫蛋白

选用苏云金芽胞杆菌CTC菌株细胞表面S 层蛋白作为表面展示载体 ,利用其N 端信号肽以及锚定序列分别将不同生物特性的外源蛋白带到胞外并锚定在细胞的表面。为研究外源蛋白的分子量和生化特性对表面展示以及对其在细胞表面生物活性的影响 ,选用分子量较大的分泌性蛋白α 淀粉酶和富含半胱氨酸的非分泌性蛋白金属硫蛋白作为目标蛋白。将α 淀粉酶的结构基因 (amy)和金属硫蛋白的结构基因 (smtA)与S 层蛋白的锚定区slh结合 ,构建融合蛋白基因slh amy及slh smtA ,克隆至穿梭载体pHT30 4上 ,得到重组质粒pBMBSA 30 4和pBMBSM 30 4 ;转入带有帮助质粒的重组菌株BMB171 pBMB CSA中 ,得到新的重组菌株BMBSAC和BMBSMC。SDS PAGE显示融合蛋白SLH AMY和SLH SMTA在重组菌的表面得到了表达。利用锥虫蓝染色 打孔加膜法表明重组菌SAC的确具有表面酶活性 ,α 淀粉酶被固定在细胞表面。碘液法测定的数据表明重组菌SAC的菌悬液样品较之受体菌BMB171菌悬液样品酶活提高了 4 2 4 %。Ni NTA 琼脂糖微球吸附试验显示重组菌SMC能够被微球所吸附 ,证实SMTA在重组菌表面具有活性。对游离镉离子的吸附试验显示重组菌对镉离子的吸附能力是对照受体菌的 4倍。     

酵母细胞表面展示技术及其在非水相酶催化合成中的应用

酵母展示技术是将外源蛋白与酵母细胞壁蛋白融合,并将外源蛋白表达在酵母细胞表面。酵母展示技术已广泛应用于各种功能蛋白的表达及筛选。以下重点介绍酵母展示技术在脂肪酶展示体系构建及其在脂肪酸甲酯、短链芳香酯及糖酯生物合成中的应用。     

重组成冰核菌适用于冷冻食品加工

为了找到关闭成冰核蛋白表达的方法.以便防止作物霜冻,已经作了大量的工作。一些集群于普通食用作物表面的好气革兰氏阴性细菌在其(菌体)表面表达成冰核蛋白。任何物质结晶都需要一个起始点,水也不例外。这些革兰氏阴性细菌的膜蛋白就提供了这样的起始点。 人们希望关闭细菌的成冰核蛋白的表达,因为冰晶的形成每年都造成食用作物的极大损失。例如,丁香假单胞菌(Pseudomonas syringae)是多种常见作物和果树的病原体,它表达inaZ成冰核基因,每年     

利用冰核蛋白N末端结构域在大肠杆菌表面展示粘质沙雷氏菌脂肪酶

【目的】利用细胞表面工程技术将活性脂肪酶展示于大肠杆菌细胞表面并对展示脂肪酶的酶学性质进行研究。【方法】将丁香假单胞菌冰核蛋白N末端结构域序列与粘质沙雷氏菌脂肪酶编码基因融合,构建成脂肪酶表面展示载体,并转化大肠杆菌BL21(DE3)。【结果】重组菌以终浓度0.05 mmol/L异丙基硫代-D-半乳糖苷(IPTG)、25°C条件下诱导培养,16 h后表面展示脂肪酶活力达到最大值1 852 U/g细胞干重。表面展示酶的最适pH为9.0,最适反应温度为40°C,表面展示酶热稳定性较游离酶有较大提高,在40°C孵育1 h后仍能保持90%以上的酶活力。【结论】以上结果表明细菌表面展示技术为脂肪酶固定提供了一个很有前景的替代方法。     

芽胞表面展示技术研究进展

芽胞表面展示技术作为微生物表面展示技术的一种,因所表达的异源蛋白无需经过跨膜过程及芽胞的抗逆性等独特优势而备受研究者的关注。以下介绍了芽胞的生理结构和形成过程、芽胞表面展示系统构建原则及目前所构建的芽胞表面展示系统种类。芽胞表面展示技术不仅在疫苗生产中应用广泛,在生物催化和细胞工厂研究领域也具有广阔的应用前景。     

两种主流载体蛋白在大肠杆菌表面展示抗体应用中的系统分析

抗体表面展示技术对于新抗体的筛选和抗体亲和力的成熟是非常重要的工具．目前,较为广泛应用的表面展示技术是噬菌体的表面展示和酵母的表面展示．大肠杆菌,以其培养简单和基因改造便捷,有望成为非常好的一种表面展示的宿主．但是,目前为止,大肠杆菌还没有被广泛地应用于抗体的表面展示技术中．主要的原因之一是在大肠杆菌外膜展示抗体的效率还不够高．作为外膜展示的载体,许多蛋白都被研究过,其中自转运蛋白(autotransporter,AT)和冰核蛋白(ice nucleation protein,INP)是人们研究最多的两种载体蛋白．还有一个原因是大肠杆菌作为宿主,在表达异源基因和展示异源蛋白过程中的存活率问题．在本研究中,系统地研究了Ag43β(一种自转运蛋白Antigen43的β结构域)和INPNC(去掉中间冗余序列的冰核蛋白的N端和C端)两种载体蛋白在强弱不同的三种启动子(T7、araBAD和lac)诱导表达的情况下,表达量、展示率、抗原亲和力以及宿主菌存活率的差异．我们发现,Ag43β展示的抗体在抗原亲和力上优于INPNC展示的抗体．在存活率方面,T7启动子诱导表达的存活率很低：用INPNC作为载体蛋白时只有0.0033%,用Ag43β作为载体蛋白时只有0.02%存活率．lac启动子诱导表达的存活率：用INPNC作为载体蛋白时为2.04%,用Ag43β作为载体蛋白时为13.27%．araBAD启动子诱导表达的存活率很高：用INPNC作为载体蛋白时为37.80%,用Ag43β作为载体蛋白时高达90.23%．但是araBAD诱导表达量和展示率都很低,所以其表现出的宿主高存活率意义有限．综合看来,由lac启动子驱动的、以Ag43?茁为载体蛋白的抗体表面展示系统是最好的选择．     

Surface Display of GFP by <Emphasis Type="Italic">Pseudomonas Syringae</Emphasis> Truncated Ice Nucleation Protein in Attenuated <Emphasis Type="Italic">Vibrio Anguillarum</Emphasis> Strain

Microbial cell surface display of foreign proteins has been widely developed for many potential applications in live vaccine construction, whole-cell biocatalysts, and bioadsorption. To investigate the feasibility of displaying heterologous proteins on the surface of attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different display systems were built upon a truncated ice nucleation protein (INP) from Pseudomonas syringae ICMP3023 whose N- and C-terminal domains were considered to be the putative membrane-anchoring motifs. Green fluorescent protein (GFP), as a reporter, was fused with the display systems in different forms of N-GFP, NC-GFP, and N-GFP-C. Analysis of the total expression level and surface localization of GFP demonstrated that the truncated P. syringae INP could be used to display foreign protein in V. anguillarum, while the system of N-GFP showed the higher levels of total expression and surface display based on unit cell density among the three and might be available for further carrier vaccine development.     

Cell surface display of salmobin, a thrombin-like enzyme from Agkistrodon halys venom on Escherichia coli using ice nucleation protein

Cell surface display on Escherichia coli using ice nucleation protein was performed in order to develop a new expression system for recombinant eukaryotic proteins. Salmobin, the thrombin-like enzyme obtained from Korean snake (Agkistrodon halys) venom was displayed on the surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. The thrombin cleavage site was inserted between salmobin and INP. The presence of salmobin on the bacterial cell surface was verified by SDS-PAGE, Western blotting, whole cell ELISA, and immunofluorescence microscopy. After thrombin cleavage the thrombin-like enzyme activity of recombinant salmobin was tested and verified. We concluded that INP-based cell surface display can be used as a novel expression system for eukaryotic proteins.     

Microbial cell-surface display

Cell-surface display allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs. The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-terminal fusion, C-terminal fusion or sandwich fusion. The characteristics of carrier protein, passenger protein and host cell, and fusion method all affect the efficiency of surface display of proteins. Microbial cell-surface display has many potential applications, including live vaccine development, peptide library screening, bioconversion using whole cell biocatalyst and bioadsorption.     

Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein

A new system designed for cell surface display of recombinant proteins on Escherichia coli was evaluated for expression of eukaryotic viral antigens. The major surface antigen of hepatitis B virus (HBsAg) was fused to the ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, whole-cell ELISA, and ice nucleation activity assay confirmed expression of recombinant proteins on the surface of Escherichia coli. This study indicated that INP-based cell surface display can be used for epitope mapping and recombinant bacteria expressing hepatitis viral antigens may be used for developing live vaccines.     

Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface

Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INP- VacII fusion protein of the expected size (52 kDa).     

Decorating microbes: surface display of proteins on Escherichia coli

Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins. By contrast, display systems based on autotransporter proteins (ATs) and ice nucleation protein (INP) are excellent systems for the display of large and complex proteins, and are therefore of considerable biotechnological relevance. Here, we review recent advances in AT and INP-mediated display and their biotechnological applications. Additionally, we discuss several promising alternative display methods, as well as novel bacterial host organisms.     

Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity

The gene encoding chitinase 92 (Chi92) from Aeromonas hydrophila JP10 has been displayed on the cell surface of Escherichia coli using the N-terminal region of ice nucleation proteins (INPN) as an anchoring motif. Immunofluorescence microscopy confirmed that Chi92 was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INPN-Chi92 fusion protein of the expected size (112 kDa). Whole cell enzyme assay indicated that the displayed Chi92 showed enhanced catalytic activity toward colloidal chitin. In addition, the Chi92-displayed cells exhibited inhibitory effects on the mycelial growth of phytopathogenic fungi, including Fusarium decemcellulare, Sclerotium rolfsii, Rhizoctonia solani kuhn, and Fusarium oxysporum f.sp. melonis. This study suggested that the INP-based display systems can be used to express a large protein (90 kDa Chi92) on the cell surface of E. coli without growth inhibition. In addition, the display of chitinase on the cell surface may provide an attractive method for the development of biocontrol agents against phytopathogenic fungi.     

Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein

A new system for cell surface display of recombinant proteins on Escherichia coli was tested for expression of the ecto domain of CD8, which is the surface protein of human T cytotoxic lymphocytes. Immunofluorescence microscopy, ELISA, and immunodot blotting confirmed successful expression of the CD8 ecto domain fused to ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. © Rapid Science Ltd. 1998     

Construction of an <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> cell-surface display system using a putative GPI-anchored protein

A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of β-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, β-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.