Surface display on staphylococci: a comparative study

Two different host-vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence-activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.     

Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display

We have investigated a staphylococcal surface display system for its potential future use as a protein library display system in combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1:1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections.     

Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display

The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.     

Protein engineering,cofactor engineering,and surface display engineering to achieve whole-cell catalytic production of chondroitin sulfate A

Chondroitin sulfate A (CSA) is a valuable glycosaminoglycan that has great market demand. However, current synthetic methods are limited by requiring the expensive sulfate group donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and inefficient enzyme carbohydrate sulfotransferase 11 (CHST11). Herein, we report the design and integration of the PAPS synthesis and sulfotransferase pathways to realize whole-cell catalytic production of CSA. Using mechanism-based protein engineering, we improved the thermostability and catalytic efficiency of CHST11; its T_m and half-life increased by 6.9°C and 3.5 h, respectively, and its specific activity increased 2.1-fold. Via cofactor engineering, we designed a dual-cycle strategy of regenerating ATP and PAPS to increase the supply of PAPS. Through surface display engineering, we realized the outer membrane expression of CHST11 and constructed a whole-cell catalytic system of CSA production with an 89.5% conversion rate. This whole-cell catalytic process provides a promising method for the industrial production of CSA.     

Bacterial display in combinatorial protein engineering

Technologies for display of recombinant protein libraries are today essential tools in many research-intensive fields, such as in the drug discovery processes of biopharmaceutical development. Phage display is still the most widely used method, but alternative systems are available and are becoming increasingly popular. The most rapidly expanding of the alternative systems are the cell display-based technologies, offering innovative strategies for selection and characterization of affinity proteins. Most investigations have focused on eukaryotic yeast for display of protein libraries, but similar systems are also being developed using prokaryotic hosts. This review summarizes the field of bacterial surface display with a strong emphasis on library applications for generation of new affinity proteins. The main focus will be on the most recent progress of the work on primarily Escherichia coli, but also on studies using a recently developed system for display on Gram-positive Staphylococcus carnosus. In addition, general strategies for combinatorial protein engineering using cell display are discussed along with the latest developments of new methodologies with comparisons to mainly phage display technology.     

Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain

The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells, since full-length proteins could be extracted and affinity-purified. Furthermore, surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization for the generation of whole-cell microbial tools for different applications will be discussed.     

一种抗革兰氏阳性致病菌新型靶酶——分选酶

许多革兰氏阳性菌的表面蛋白是经过一种被称为分选酶的半胱氨酸转肽酶的作用而锚定到细胞壁上,由于表面蛋白在病原菌的致病性方面起关键作用,分选酶有可能成为降低革兰氏阳性菌致病性的药物靶点.目前,通过对金黄色葡萄球菌中分选酶A(SrtA)的研究,已初步阐明分选酶的作用机制及其活性位点,与此同时,一些SrtA抑制剂的初步鉴定为今后抑制剂更深层次的筛选提供了基础.     

An effective peptide screening system using recombinant fluorescent bacterial surface display

An effective method for peptide screening of ligand-binding proteins was applied by using recombinant E. coli which is capable of expressing green fluorescent protein (GFP) and which can also express random peptides displayed on flagella of the cells. This screening method used a combination of fluorescence-activated cell sorting (FACS) and flagella display on the basis of a commercial FliTrx random peptide library for isolating the peptide-displaying clones which are able to bind Alexa 546 fluorescence-labeled cytochrome c. Flow cytometry simultaneously detected the two different fluorescence intensities, from GFP in the library and Alexa546-labeled to cytochrome c, enabling the specific clones bound to cytochrome c to be obtained from the first or second round of cell sorting. Compared with original FliTrx peptide screening system that requires repeating biopanning five times, our results suggested that detecting two different fluorescence intensities by flow cytometry is feasible for effective peptide screening.     

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.     

金黄色葡萄球菌表面蛋白研究进展

金黄色葡萄球菌是一种常见的人兽共患病的病原菌。通过其表面蛋白(黏附素)与宿主的细胞外基质结合感染宿主,这些蛋白的结构已从分子水平上得到揭示。本综述了金葡菌产生的表面蛋白及其主要蛋白的分子结构。     

鼠李糖乳杆菌颗粒表面展示系统的构建

为了比较不同锚钩蛋白基序结合活性,构建新型的鼠李糖乳杆菌颗粒表面展示系统。首先,用热酸处理法制备鼠李糖乳杆菌GEM(Gram-positive enhancer matrix,GEM)颗粒,并通过电镜观察、RT-PCR检测和SDS-PAGE检测鉴定其处理效果;同时,利用大肠杆菌表达了锚定蛋白PA3-EGFP和P60-EGFP并将其与GEM颗粒共同孵育结合;最后,使用免疫印迹、电镜观察、荧光显微镜观察和荧光分光光度法评价鼠李糖乳杆菌GEM颗粒与锚定蛋白的结合效率。结果表明,使用10%的TCA处理鼠李糖乳杆菌得到了灭活的肽聚糖骨架(GEM颗粒),经鉴定其大小形态均一,绝大部分无蛋白残留,3.8×10~6个GEM颗粒样品中的DNA拷贝数仅为32;免疫印迹和荧光显微镜观察均可检测到融合蛋白PA3-EGFP和P60-EGFP锚定在GEM颗粒上,且结合在GEM颗粒表面的锚定蛋白呈絮状。荧光分光光度计法检测结果显示锚定蛋白PA3-EGFP结合GEM的效率稍高于P60-EGFP,但差异不显著(P0.05)。以上结果表明由鼠李糖乳杆菌制得的GEM颗粒与锚定蛋白PA3、P60的结合效率良好,可用于构建新型的外源蛋白表面展示系统,进而为后续的细菌样颗粒疫苗的研究与应用奠定基础。     

丝状真菌表面展示技术研究进展

丝状真菌表面展示技术是将表达的目的蛋白固定在丝状真菌细胞表面的一项新兴基因工程技术。丝状真菌具有极强的蛋白质分泌能力和良好的蛋白质翻译后加工能力,因而越来越多的丝状真菌表面展示技术得到开发和应用。本文就丝状真菌表面展示系统的研发和应用进展进行综述,并介绍与该系统构建密切相关的丝状真菌的细胞壁组成、锚定蛋白和遗传转化方法等技术。     

A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping

Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full‐length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full‐length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark‐derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A‐GFP system. Yeast library screening of full‐length vNAR variants which were detected via GFP expression yielded the same high‐affinity binder that had previously been isolated by our group using the conventional epitope tag‐based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost‐friendly alternative to conventional epitope tag detections.     

Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface

Cell surface display was used as a strategy to display the gold-binding polypeptide (GBP) fusion protein on the surface of Escherichia coli , and consequently to immobilize the cells on the gold surface. The DNA encoding the GBP was fused to the truncated fadL gene and was expressed by the tac promoter. For the display of the core streptavidin (cSA) of Streptomyces avidinii , the cSA gene was fused to the truncated oprF gene. After the dual display of FadL–GBP and OprF–cSA on the surface of E. coli , binding of cells on the gold surface and the interaction of OprF–cSA with the biotin–horseradish peroxidase (HRP) were studied by surface plasmon resonance (SPR) analysis. Cells displaying the FadL–GBP fusion protein could be immobilized on the SPR sensor chip as shown by the SPR angle shift of 0.5°, which was stably bound at least for 60 h with a washing solution. When the FadL–GBP and OprF–cSA fusion proteins were displayed on the same cell surface, the former was used to immobilize the cells on the gold surface and the latter was used for the interaction studies with the biotin–HRP, which demonstrates that the strategy should be useful for developing whole-cell biosensor chips.     

Anchoring of proteins to lactic acid bacteria

The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.     

噬菌体表面呈现技术研究进展

噬菌体表面呈现技术是1985年建立的一种将外源基因表达呈现在噬菌体颗粒表面的方法,可用于建立随机多肽文库、抗体文库等。经特定配基的筛选,可获得与其特异结合的配体分子。通过改构,还可将cDNA产物表达于噬菌体颗粒的尾部构建cDNA文库。SIP技术通过将配体和配基分别与基因Ⅲ蛋白的C末端和N末端融合表达,基因Ⅲ的C-末端参与噬菌体颗粒的组装,配基与配体的结合能够重建基因Ⅲ蛋白的功能,才能形成有感染能力的噬菌体,这样就大大提高了筛选效率。     

Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism

AIMS: The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries. METHODS AND RESULTS: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population. CONCLUSIONS: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation. SIGNIFICANCE AND IMPACT OF THE STUDY: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.     

Bacterial cell surface display for epitope mapping of hepatitis C virus core antigen

Cell surface expression of protein has been widely used to display enzymes and antigens. Here we show that Pseudomonas syringae ice nucleation protein with a deletion of internal repeating domain (INC) can be used in Escherichia coli to display peptide in a conformationally active form on the outside of the folded protein by fusing to the C-terminus of INC. Diagnostic potential of this technology was demonstrated by effective mapping of antigenic epitopes derived from hepatitis C virus (HCV) core protein. Amino acids 1-38 and 26-53 of HCV core protein were found to react more sensitively in a native conformation with the HCV patient sera than commercial diagnostic antigen, c22p (amino acids 10-53) by display-ELISA. These results demonstrate that the bacterial cell surface display using INC is useful for peptide presentation and thus epitope mapping of antigen.     

基于蛋白质跨外膜自转运系统的细菌细胞表面蛋白展示技术研究进展

革兰氏阴性菌Ⅴ型分泌系统是细菌病原蛋白分泌的主要途径之一,可分为Ⅴa-Ⅴe5个亚型,其中Ⅴa型(即经典的单体自转运蛋白)是细菌毒力和黏附因子向细胞外分泌的重要工具,其在内膜Sec易位子和外膜BAM蛋白复合体的协助下,通过2个连续的跨膜步骤介导蛋白质穿过阴性菌的内外膜.据信Va型是目前已知蛋白质跨膜转运时最简单的分泌途径...     

Surface display of a glucose binding protein

Glucose binding protein (GBP) from Escherichia coli has been widely used to develop minimally invasive glucose biosensors for diabetics. To develop a cell-based glucose biosensor, it is essential to functionally display GBP on the cell surface. In this study, we designed a molecular structure to display GBP on the outer membrane of E. coli. We fused GBP with the first nine N-terminal residues of Lpp (major E. coli lipoprotein) and the 46–150 residues of OmpA (an outer membrane protein of E. coli). With this molecular design, we have successfully displayed GBP on the surface of E. coli. Using FITC-conjugated Dextran, we demonstrated that glucose’s binding sites of surface-displayed GBP were accessible to glucose. Furthermore, we showed that glucose transport in a GBP-deficient E. coli NM303 could be restored by displaying GBP on the surface of NM303. 0.51 h⁻¹ of specific growth rate was attained for NM303/pESDG grown in M9 minimal medium supplemented with 2 g/l glucose, whereas no growth was observed for NM303 in the same medium. Both NM303 and NM303/pESDG grew in M9 medium supplemented with 1 mM of fucose. Because cell surface is an interface between intracellular and extracellular molecular events, this technique paves a way to develop cell-based glucose biosensors.