Display Of Recombinant Proteins Research Articles

Genome-Scale Mining of Novel Anchor Proteins of Corynebacterium glutamicum.

The display of recombinant proteins on the surfaces of bacteria is a research topic with many possible biotechnology applications—among which, the choice of host cell and anchoring motif is the key for efficient display. Corynebacterium glutamicum is a promising host for surface display due to its natural advantages, while single screening methods and fewer anchor proteins restrict its application. In this study, the subcellular localization (SCL) predictor LocateP and tied-mixture hidden Markov models were used to analyze all five known endogenous anchor proteins of C. glutamicum and test the accuracy of the predictions. Using these two tools, the SCLs of all proteins encoded by the genome of C. glutamicum 13032 were predicted, and 14 potential anchor proteins were screened. Compared with the positive controls NCgl1221 and NCgl1337, three anchoring proteins—NCgl1307, NCgl2775, and NCgl0717—performed better. This study also discussed the applicability of the anchor protein screening method used in this experiment to other bacteria.

Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems

BackgroundThe display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins.ResultsIn this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp’OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp’OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy.ConclusionsTo our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp’OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.

Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli

BackgroundPhthalic acid esters (PAEs) are widely used as plasticizers or additives during the industrial manufacturing of plastic products. PAEs have been detected in both aquatic and terrestrial environments due to their overuse. Exposure of PAEs results in human health concerns and environmental pollution. Diisobutyl phthalate is one of the main plasticizers in PAEs. Cell surface display of recombinant proteins has become a powerful tool for biotechnology applications. In this current study, a carboxylesterase was displayed on the surface of Escherichia coli cells, for use as whole-cell biocatalyst in diisobutyl phthalate biodegradation.ResultsA carboxylesterase-encoding gene (carEW) identified from Bacillus sp. K91, was fused to the N-terminal of ice nucleation protein (inpn) anchor from Pseudomonas syringae and gfp gene, and the fused protein was then cloned into pET-28a(+) vector and was expressed in Escherichia coli BL21(DE3) cells. The surface localization of INPN-CarEW/or INPN-CarEW-GFP fusion protein was confirmed by SDS-PAGE, western blot, proteinase accessibility assay, and green fluorescence measurement. The catalytic activity of the constructed E. coli surface-displayed cells was determined. The cell-surface-displayed CarEW displayed optimal temperature of 45 °C and optimal pH of 9.0, using p-NPC2 as substrate. In addition, the whole cell biocatalyst retained ~ 100% and ~ 200% of its original activity per OD600 over a period of 23 days at 45 °C and one month at 4 °C, exhibiting the better stability than free CarEW. Furthermore, approximately 1.5 mg/ml of DiBP was degraded by 10 U of surface-displayed CarEW cells in 120 min.ConclusionsThis work provides a promising strategy of cost-efficient biodegradation of diisobutyl phthalate for environmental bioremediation by displaying CarEW on the surface of E. coli cells. This approach might also provide a reference in treatment of other different kinds of environmental pollutants by displaying the enzyme of interest on the cell surface of a harmless microorganism.

Screening for New Surface Anchoring Domains for Lactococcus lactis.

The display of recombinant proteins on bacterial surfaces is a developing research area with a wide range of potential biotechnological applications. The lactic acid bacterium Lactococcus lactis is an attractive host for such surface display, and a promising vector for in vivo delivery of bioactive proteins. Surface-displayed recombinant proteins are usually anchored to the bacterial cell wall through anchoring domains. Here, we investigated alternatives to the commonly applied lactococcal lysine motif (LysM)-containing surface anchoring domain, the C-terminus of AcmA (cAcmA). We screened 15 anchoring domains of lactococcal or phage origins that belong to the Pfam categories LPXTG, LysM, CW_1, Cpl-7, WxL, SH3, and ChW, which can provide non-covalent or covalent binding to the cell wall. LPXTG, LysM, the duplicated CW_1 and SH3 domains promoted significant surface display of two model proteins, B domain and DARPin I07, although the display achieved was lower than that for the reference anchoring domain, cAcmA. On the other hand, the ChW-containing anchoring domain of the lactococcal phage AM12 endolysin (cAM12) demonstrated surface display comparable to that of cAcmA. The anchoring ability of cAM12 was confirmed by enabling non-covalent heterologous anchoring of the B domain on wild-type bacteria, as well as anchoring of CXCL8-binding evasin-3, which provided potential therapeutic applicability; both were displayed to an extent comparable to that of cAcmA. We have thereby demonstrated the effective use of different protein anchoring domains in L. lactis, with ChW-containing cAM12 the most promising alternative to the established approaches for surface display on L. lactis.

Surface Anchoring on Lactococcus lactis by Covalent Isopeptide Bond

Display of recombinant proteins on the bacterial surface is an emerging research area with wide range of potential biotechnological applications. Because of its GRAS (generally recognized as safe) status, lactic acid bacterium Lactococcus lactis represents an attractive host for surface display and promising vector for in situ delivery of bioactive proteins. The present study focused on finding a new alternative approach for surface display on Lactoccocus lactis. We developed a system that enables the formation of irreversible isopeptide bonds on the surface of Lactococus lactis. This was achieved through the following two protein/peptide pairs, SpyCatcher/SpyTag and SnoopCatcher/SnoopTag.1-3 Attachment of tagged model protein B domain to the cell surface of Lactococccus lactis displaying the corresponding catcher protein was demonstrated using flow cytometry. We demonstrated effective use of aforementioned protein anchors which thus represent a promising alternative to established approaches for surface display on Lactoccocus lactis.

Engineering E. coli cell surface in order to develop a one-step purification method for recombinant proteins

Sortases are enzymes mostly found in Gram-positive bacteria which cleave proteins site-specifically. This feature makes them a promising tool in molecular biology and biotechnology. In this study, using bacterial surface display of recombinant proteins and ability of sortase A in site-specifically cleavage of the amino acid sequences, a novel method for one-step purification of recombinant proteins was developed. Using computational program tools, a chimeric protein containing a metallothionein (mt) and chitin binding domain (ChBD) was attached to the C-terminal domain of the truncated outer membrane protein A (Lpp′-ompA) using sortase recognition site (amino acid residues: LPQTG) as a separator. The structure of the chimeric protein was simulated using molecular dynamics to determine if the LPQTG motif is accessible to the sortase active site. The designed chimeric protein was expressed and purified. The purified chimeric protein was also displayed on the surface of E. coli cells. Both purified chimeric protein and the E. coli cells displaying Lpp′-ompA-mt-ChBD carrier protein were then treated with sortase to evaluate the efficiency of sortase-mediated cleavage of purified chimeric protein as well as surface displayed-chimeric protein. It is shown that mt-ChBD protein was successfully cleaved and dissociated from Lpp′-ompA carrier and released into the medium after treatment with sortase in both recombinant protein and surface displayed-chimeric protein. The experimental results confirmed the molecular dynamics analysis results. The presented method could be regarded as a novel strategy for one step expression and purification of recombinant proteins.

Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation

The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications.

Lactic acid bacteria (LAB) are promising vectors of choice to deliver active molecules to mucosal tissues. They are recognized as safe by the World Health Organization and some strains have probiotic properties. The wide range of potential applications of LAB-driven mucosal delivery includes control of inflammatory bowel disease, vaccine delivery, and management of auto-immune diseases. Because of this potential, strategies for the display of proteins at the surface of LAB are gaining interest. To display a protein at the surface of LAB, a signal peptide and an anchor domain are necessary. The recombinant protein can be attached to the membrane layer, using a transmembrane anchor or a lipoprotein-anchor, or to the cell wall, by a covalent link using sortase mediated anchoring via the LPXTG motif, or by non-covalent liaisons employing binding domains such as LysM or WxL. Both the stability and functionality of the displayed proteins will be affected by the kind of anchor used. The most commonly surfaced exposed recombinant proteins produced in LAB are antigens and antibodies and the most commonly used LAB are lactococci and lactobacilli. Although it is not necessarily so that surface-display is the preferred localization in all cases, it has been shown that for certain applications, such as delivery of the human papillomavirus E7 antigen, surface-display elicits better biological responses, compared to cytosolic expression or secretion. Recent developments include the display of peptides and proteins targeting host cell receptors, for the purpose of enhancing the interactions between LAB and host. Surface-display technologies have other potential applications, such as degradation of biomass, which is of importance for some potential industrial applications of LAB.

A structurally informed autotransporter platform for efficient heterologous protein secretion and display

BackgroundThe self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein.ResultsBased on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development.ConclusionsWe developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.

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

Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the expression of fully functional eukaryotic proteins inE. coli. Essential to the success of these procedures is the transport of such proteins across the inner membrane to the periplasmic space, allowing proper folding and the establishment of disulfide bonding. Subsequently, fully functional proteins can be exposed on the surface of filamentous (bacterio)phages, provided a system is employed that consists of a cloning vector (e.g. the phagemid pComb3, Barbas et al., 1991) that generates phage particles in the presence of a helper phage. The main advantage of surface display of recombinant proteins is to facilitate the screening of very large numbers of different molecules by simple selection methods ("panning"). In addition, periplasmic expression yields relatively large quantities (e.g. 1 mg l(-1) of culture) soluble protein. In this review, the principle aspects of this novel expression system based on the phagemid pComb3 will be discussed. Two examples for functional periplasmic expression of human proteins inE. coli will be presented, namely i) the antigen-binding moiety (Fab fragment) of human immunoglobulins (IgGs) and ii) the human plasminogen activator inhibitor 1, an essential regulator of the plasminogen activation system. Finally, perspectives for the application of this system to express mutant proteins, fragments of proteins and peptides are indicated.