Cell Surface Display System Research Articles

Novel surface display system for heterogonous proteins on Lactobacillus plantarum

To establish a novel cell surface display system that would enable the display of target proteins on Lactobacillus plantarum. BlastP analysis of the amino acids sequence data revealed that the N-terminus of the putative muropeptidase MurO from L. plantarum contained two putative lysin motif (LysM) repeat regions, implying that the MurO was involved in bacterial cell wall binding. To investigate the potential of MurO for surface display, green fluorescent protein (GFP) was fused to MurO at its C-terminus and the resulting fusion protein was expressed in Escherichia coli. After being mixed with L. plantarum cells in vitro, GFP was successfully displayed on the surfaces of L. plantarum cells. Increases in the fluorescence intensities of chemically pretreated L. plantarum cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for MurO. SDS sensitivity assay showed that the GFP fluorescence intensity was reduced after being treated with SDS. To demonstrate the applicability of the MurO-mediated surface display system, β-galactosidase from Bifidobacterium bifidium, in place of GFP, was functionally displayed on the surface of L. plantarum cells via MurO. The MurO was a novel anchor protein for constructing a surface display system for L. plantarum. The success in surface display of GFP and β-galactosidase opened up the feasibility of employing the cell wall anchor of MurO for surface display in L. plantarum.

Immobilization of recombinant nanobiofiber CS3 fimbriae onto alginate beads for improvement of cadmium biosorption

A cell surface display system with metalbinding properties was previously developed using CS3 fimbriae, which are hollow tubes 20 nm-thick and 2 nm in diameter. In this study, hybrid CS3 pili were separated from recombinant Escherichia coli and entrapped in calcium alginate gel beads in order to improve their stabilization and also adsorption of heavy metals. The surface morphology of the gel beads containing pili was investigated by scanning electron microscopy (SEM). Immunofluorescence microscopy was employed to confirm the attachment of nanobiofibers to the alginate beads. The effects of three variables (sodium alginate concentration, protein to alginate mass ratio, and bead size) at two levels each on Cd2+ biosorption efficiency were investigated by full factorial experimental design. A second-order polynomial equation modeled the design space for the process response of cadmium removal capacity. The optimal values of the factors were obtained as follows: 1% sodium alginate concentration, 0.25 protein to alginate mass ratio, and a 6 mm bead size. Under these conditions, Cd2+ was adsorbed at 45.45 mg/g to the nanobiofiber. The results indicate that the immobilized recombinant hybrid CS3 pili may be an appropriate biosorbent for removal of heavy metals from polluted aquatic environments.

Construction of Copper Removing Bacteria Through the Integration of Two-Component System and Cell Surface Display

Synthetic biological systems are becoming more and more feasible for commercial and medical purposes through the genetic engineering of several components. The simple assembly of a genetic circuit was shown to stimulate the removal of copper by bacteria through the engineering of a two-component system. The CusSR two-component systems is a regulator of Escherichia coli copper homeostatic system. In this system, genetic circuits of CusSR were fused to a cell surface display system for metal adsorption; this system is suitable for the display of a copper binding peptide through outer membrane protein C (OmpC). E. coli ompC codes for an outer membrane pore protein (porin) are induced at high osmolarity and temperature, which can also be used as an anchoring motif to accept the passenger proteins. The bacteria that produce the chimeric OmpC containing the copper binding peptide adsorbed maximum concentrations of 92.2 μmol of Cu(2+)/gram dry weight of bacterial cells. This synthetic bacterial system senses the specific heavy metal and activates a cell surface display system that acts to remove the metal.

Bacterial Display and Screening of Posttranslationally Thioether-Stabilized Peptides

A major hurdle in the application of therapeutic peptides is their rapid degradation by peptidases. Thioether bridges effectively protect therapeutic peptides against breakdown, thereby strongly increasing bioavailability, enabling oral and pulmonary delivery and potentially significantly optimizing the receptor interaction of selected variants. To efficiently select optimal variants, a library of DNA-coupled thioether-bridged peptides is highly desirable. Here, we present a unique cell surface display system of thioether-bridged peptides and successfully demonstrate highly selective screening. Peptides are posttranslationally modified by thioether bridge-installing enzymes in Lactococcus lactis, followed by export and sortase-mediated covalent coupling to the lactococcal cell wall. This allows the combinatorial optimization and selection of medically and economically highly important therapeutic peptides with strongly enhanced therapeutic potential.

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

A study on the dynamics of the zraP gene expression profile and its application to the construction of zinc adsorption bacteria

Zinc ion plays essential roles in biological chemistry. Bacteria acquire Zn(2+) from the environment, and cellular concentration levels are controlled by zinc homeostasis systems. In comparison with other homeostatic systems, the ZraSR two-component system was found to be more efficient in responding to exogenous zinc concentrations. To understand the dynamic response of the bacterium ZraSR two-component system with respect to exogenous zinc concentrations, the genetic circuit of the ZraSR system was integrated with a reporter protein. This study was helpful in the construction of an E. coli system that can display selective metal binding peptides on the surface of the cell in response to exogenous zinc. The engineered bacterial system for monitoring exogenous zinc was successfully employed to detect levels of zinc as low as 0.001 mM, which directly activates the expression of chimeric ompC(t)--zinc binding peptide gene to remove zinc by adsorbing a maximum of 163.6 μmol of zinc per gram of dry cell weight. These results indicate that the engineered bacterial strain developed in the present study can sense the specific heavy metal and activates a cell surface display system that acts to remove the metal.

In vivo and in vitro surface display of heterologous proteins on Bacillus thuringiensis vegetative cells and spores

The Bacillus surface display system is a heat-stable platform for heterologous protein expression that preserves biological activity in an adverse environment. In the present study, a new cell surface display system that enables heterologous proteins to be displayed consecutively on the surface of Bacillus thuringiensis vegetative cells and spores in vivo and in vitro is reported. Using the N-terminal domain (Mbgn) of a peptidoglycan hydrolase (Mbg) as anchor, the green fluorescence protein (GFP) and a bacterial laccase (WlacD) can be targeted onto the surface of vegetative cells and spores in vivo in an active form. This was confirmed by GFP fluorescence intensity quantification, enzymatic activity measurement, immunofluorescence microscopy examination, and flow cytometry assay. Immobilization of externally added Mbgn-mediated fusion proteins in vitro to obtain actively displaying GFP and/or WlacD was subsequently achieved in B. thuringiensis, Bacillus subtilis and Bacillus cereus, demonstrating the versatility of the proposed system. Therefore, Mbgn-mediated in vivo and in vitro immobilization of foreign proteins represents an improved method of microbial display for extended biotechnological applications.

Secretion-and-capture cell-surface display for selection of target-binding proteins

This report describes a new cell-surface display system, the Secretion and Capture Technology (SECANT™) platform, which relies on in vivo biotinylation of the protein of interest followed by its capture on the avidinated surface of the parent cell. Cell sorting techniques are then used to isolate clones that display target-binding protein. A distinguishing feature of this method is its ability to display complex proteins, such as full-length immunoglobulin G (IgG) antibodies, on living cells. In this proof-of-concept study, Saccharomyces cerevisiae cells that displayed Herceptin IgG were isolated from a 10,000-fold excess of cells that displayed a lysozyme-binding antibody.

Surface display of human lactoferrin using a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae in Pichia pastoris

A cell surface display system was developed in Pichia pastoris using the gene TIP1, encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae (ScTIP). Human lactoferrin cDNA (hLf) was fused to a full-length TIP1 DNA (ScTIP ( 630 )) or a short-TIP1 fragment (ScTIP ( 120 )) encoding the 40 C-terminal amino acids of ScTIP. Both hLf-ScTIP fusion genes were expressed in P. pastoris SMD 1168. The fused protein was detected by western blotting after extraction of the lysed recombinant cells with Triton X-100, urea, and Triton X-100 plus urea, suggesting that the hLf is associated with the membrane. The localization of surface-displayed hLf was confirmed by immunofluorescence confocal microscopy and flow cytometric analysis using FITC-labeled anti-hLf antibody, suggesting that hLf was successfully located at the surface of P. pastoris. The intact recombinant cells and cell lysates showed antibacterial activity against target microorganisms, meaning that the expressed hLf was biologically active. The results indicated that the ScTIP anchoring motif is useful for cell surface display of foreign proteins in P. pastoris.

High-throughput screening of improved protease inhibitors using a yeast cell surface display system and a yeast cell chip

Protease-targeted inhibitors have been promising pharmaceuticals. Here, we combined a yeast cell surface display system with a yeast cell chip for the high-throughput screening of protease inhibitors, and succeeded in improving the activity of a protease inhibitor.

Directed evolution of CotA laccase for increased substrate specificity using Bacillus subtilis spores

Directed evolution is an effective strategy to engineer and optimize protein properties, and microbial cell-surface display is a successful method to screen protein libraries. Protein surface display on Bacillus subtilis spores is demonstrated as a tool for screening protein libraries for the first time. Spore display offers advantages over more commonly utilized microbe cell-surface display systems, which include gram-negative bacteria, phage and yeast. For instance, protein-folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes; hence, the displayed proteins are "preimmobilized" on the inherently inert spore surface. Immobilized proteins have several advantages when used in industrial processes. The protein stability is increased and separations are simplified. Finally, immobilized proteins can be used in a wide array of simple device applications and configurations. The substrate specificity of the enzyme CotA is narrowed. CotA is a laccase and it occurs naturally on the outer coat of B. subtilis spores. A library of CotA genes were expressed in the spore coat, and it was screened for activity toward ABTS [diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)] over SGZ (4-hydroxy-3,5-dimethoxy-benzaldehyde azine). A mutant CotA was found to be 120-fold more specific for ABTS. This research demonstrates that B. subtilis spores can be a useful platform for screen protein libraries.

Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability

A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60 degrees C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.

Engineering of microorganisms towards recovery of rare metal ions

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved.

Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein

A Pichia pastoris cell-surface display system was constructed using the Sed1 anchor system that has been developed in Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was used as the model protein and was fused to an anchor that consisted of 338 amino acids of Sed1. The resulting fusion protein CALBSed1 was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). Immunofluorescence microscopy of immunolabeled Pichia pastoris revealed that CALB was displayed on the cell surface. Western blot analysis showed that the fusion protein CALBSed1 was attached covalently to the cell wall and was highly glycosylated. The hydrolytic activity of the displayed CALB was more than 220 U/g dry cells after 120 h of culture. The displayed protein also exhibited a higher degree of thermostability than free CALB.

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the alpha-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins alpha-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with alpha-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with alpha-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using alpha-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with alpha-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.

Enhanced Display of Lipase on the Escherichia coli Cell Surface, Based on Transcriptome Analysis

A cell surface display system was developed using Escherichia coli OmpC as an anchoring motif. The fused Pseudomonas fluorescens SIK W1 lipase was successfully displayed on the surface of E. coli cells, and the lipase activity could be enhanced by the coexpression of the gadBC genes identified by transcriptome analysis.

Molecular design of yeast cell surface for adsorption and recovery of molybdenum, one of rare metals

In modern industrial society, molybdenum is one of the important metals for development of the industry of rare metals. It is important to recycle the rare metals from wastes because they are technically and economically difficult to be dug and be purified, and they exist in only a few regions in the world. In this study, ModE protein derived from Escherichia coli, which is a molybdate-dependent transcriptional regulator with the ability to bind molybdate as a form of soluble molybdenum, was displayed on the yeast cell surface by alpha-agglutinin-based cell surface display system for the adsorption and recovery of molybdate. Displayed ModE, confirmed by immunofluorescence labeling, caught molybdate more preferably at pH 3.0 than at basic pH. Yeast cells displaying C-terminal domain of ModE, which lacks N-terminal DNA binding domain, more effectively adsorbed molybdate than those displaying full-length ModE, suggesting that the deletion of the domain unrelated to metal binding enhanced the binding ability. Our results indicated that the adsorption system on cell surface of yeast cells displaying ModE is effective not only for adsorption of molybdate as a rare metal bioadsorbent but also for the easy recovery of molybdate located on the cell surface.

Development of novel cell-surface display system of Aspergillus oryzae using chitin binding domain

Development of novel cell-surface display system of Aspergillus oryzae using chitin binding domain

Displaying non-natural, functional molecules on yeast surfaces via biotin–streptavidin interaction

Here we expand the yeast cell surface display system to display non-natural, functional molecules. The short biotin acceptor peptide (BAP) sequence of biotin ligase from E. coli (BirA) was genetically introduced to the N-terminus of the anchor protein, Flo428. Through co-expression of BAP-fused Flo428 with BirA, biotinylated BAP could be displayed on the yeast cell surface. Subsequent addition of streptavidin–FITC resulted in the display of streptavidin–FITC, and, the display of biotin–FITC was successful using streptavidin as a linker. Our strategy provides a powerful tool for displaying functional molecules on yeast cell surfaces.