Biotechnological applications for surface‐engineered bacteria

Wernérus, Henrik; Ståhl, Stefan

doi:10.1042/ba20040014

Cited by 93 publications

(19 citation statements)

References 204 publications

(283 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This technology is of interest for several applications, including live vaccine development, bioremediation through adsorption to expressed binder proteins, library screening and the development of whole cell biocatalysts [1,2]. …”

Section: Introductionmentioning

confidence: 99%

Optimisation of surface expression using the AIDA autotransporter

2011

View full text Add to dashboard Cite

BackgroundBacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.ResultsThe use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface-anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l-1 could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.ConclusionsA number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

show abstract

Section: Introductionmentioning

confidence: 99%

Optimisation of surface expression using the AIDA autotransporter

2011

View full text Add to dashboard Cite

show abstract

“…Bacterial display of proteins on the cell surface using recombinant DNA technology has been used in microbiology, biotechnology and vaccine technology and has been an area of intense research since reports of this novel technology were published [1-5]. One of the most studied areas of application has been live vaccine delivery through the surface display of antigenic proteins [2,4].…”

Section: Introductionmentioning

confidence: 99%

“…Several surface display systems for both gram negative and gram positive bacteria have been described in the literature but the detailed knowledge of these systems varies considerably [5]. Many naturally occurring proteins have been developed as carriers for a target protein, such as outer membrane proteins, lipoproteins, secreted proteins and subunits of surface appendages.…”

Section: Introductionmentioning

confidence: 99%

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus

et al. 2011

View full text Add to dashboard Cite

BackgroundSalmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.ResultsBoth SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis.ConclusionApart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

show abstract

“…Protein-displaying bacteria may be valuable biotechnological tools (Lee, Choi et al 2003, Wernerus and Stahl 2004, Schuurmann, Quehl et al 2014, Li and Tao 2015, Smith, Khera et al 2015). Several research groups have engineered vegetative B. subtilis cells to display heterologous proteins because there are well-established methods to manipulate its genome, it is used industrially to produce biocommodities, it can secrete prodigious amounts of protein, and because it has generally recognized as safe status (Schallmey, Singh et al 2004, Liu, Liu et al 2013).…”

Section: Discussionmentioning

confidence: 99%

“…Bacteria engineered to display heterologous proteins have many potential biotechnological applications, including uses in protein engineering, biocatalysis, lignocellulose degradation, biosensing and bioremediation (Lee, Choi et al 2003, Wernerus and Stahl 2004, Schuurmann, Quehl et al 2014, Li and Tao 2015, Smith, Khera et al 2015). Surface display methods are attractive because the microbe produces and displays the protein during growth, circumventing the need for time-consuming protein purification and immobilization procedures that are typically used to create protein coated materials (Garcia-Galan, Berenguer-Murcia et al 2011, Mohamad, Marzuki et al 2015, Hyeon, Shin et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Huang

Gosschalk

Kim

et al. 2018

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. To gain insight into the factors that affect displayed protein stability and copy-number, we systematically compared the ability of different protease-deficient B. subtilis strains (WB800, BRB07, BRB08 and BRB14) to display a Cel8A-LysM reporter protein in which the Clostridium thermocellum Cel8A endoglucanase is fused to LysM cell wall binding modules. Whole-cell cellulase measurements and fractionation experiments demonstrate that genetically eliminating extracytoplasmic bacterial proteases improves Cel8A-LysM displays levels. However, upon entering stationary phase, for all protease-deficient strains, the amount of displayed reporter dramatically decreases, presumably as a result of cellular autolysis. This problem can be partially overcome by adding chemical protease inhibitors, which significantly increase protein display levels. We conclude that strain BRB08 is well-suited for stably displaying our reporter protein, as genetic removal of its extracellular and cell wall-associated proteases leads to the highest levels of surface accumulated Cel8A-LysM without causing secretion stress or impairing growth. A two-step procedure is presented that enables the construction of enzyme coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3 days. The results of this work could aid the development of whole cell display systems that have useful biotechnological applications.

show abstract

Biotechnological applications for surface‐engineered bacteria

Cited by 93 publications

References 204 publications

Optimisation of surface expression using the AIDA autotransporter

Optimisation of surface expression using the AIDA autotransporter

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Contact Info

Product

Resources

About