Cell surface display of recombinant proteins on Staphylococcus carnosus

Samuelson, Patrik; Hansson, Magnus; Ahlborg, Niklas; Andréoni, C.; Götz, Friedrich; Bächi, Thomas; Nguyen, Thien Ngoc; Binz, Hans; Uhlén, Mathias; Ståhl, Stefan

doi:10.1128/jb.177.6.1470-1476.1995

Cited by 120 publications

(213 citation statements)

References 44 publications

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“…In accordance with earlier observations for proteins containing the lipase propeptide, the recovered cell wall proteins all migrated as somewhat larger than their calculated theoretical values (12,42). The wild-type strain does not express any serum albumin-binding surface protein (42). These results demonstrate that the hybrid receptors were properly expressed and localized to the cell wall of the recombinant S. carnosus cells.…”

Section: Resultssupporting

confidence: 77%

“…Full-length proteins with little or no proteolytic degradation could be recovered from the cell wall fractions of the recombinant staphylococci. In accordance with earlier observations for proteins containing the lipase propeptide, the recovered cell wall proteins all migrated as somewhat larger than their calculated theoretical values (12,42). The wild-type strain does not express any serum albumin-binding surface protein (42).…”

Section: Resultssupporting

confidence: 72%

“…The ABP domain is expressed as the part of the chimeric surface protein closest to the cell wall-anchoring regions. This ABP region has proven to be useful as a reporter molecule in a colorimetric assay to analyze the surface accessibility of the expressed chimeric surface proteins (42), as an affinity tag for recovery of recombinant surface proteins (42), and also as a spacer protein to increase the surface accessibility of displayed proteins (50). Note that the PP from S. hyicus is not processed in S. carnosus (12), while it is cleaved off in its homologous host S. hyicus (1,2).…”

Section: Resultsmentioning

confidence: 99%

“…MЈ represents the processed and covalently anchored form of the M sequence of S. aureus protein A (29,45). Also present in the vector is a multifunctional albumin-binding protein (ABP) derived from streptococcal protein G (34,42). The ABP domain is expressed as the part of the chimeric surface protein closest to the cell wall-anchoring regions.…”

Section: Resultsmentioning

confidence: 99%

“…A gram-positive bacterium that has been investigated extensively for various surface display applications is the nonpathogenic S. carnosus (26,42) which is used traditionally in starter cultures in meat fermentation applications (18). Recombinant S. carnosus strains with various proteins expressed on the surface have been successfully evaluated as live bacterial vaccine delivery vehicles (5,6,50), for potential diagnostic applications through the display of single-chain Fv antibody fragments (16) and engineered S. aureus protein A domains, called affibodies (15).…”

mentioning

confidence: 99%

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Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Wernérus

Lehtiö

Teeri

et al. 2001

Appl Environ Microbiol

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Ni2؉ -binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni 2؉ -binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni 2؉ -binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.Bacterial surface display of heterologous proteins has in recent years become an increasingly active research area with a wide range of applications in immunology, vaccinology, and biotechnology (11, 49). An interesting application area is the display of metal-binding proteins on bacteria to create wholecell tools for improved sequestration of toxic metals in wastewater (3), a field in which naturally occurring bacteria have been evaluated previously (10, 31). The heterologous expression of peptides and proteins with inherent metal-binding capacity have been employed to create bacteria with improved metalloadsorption properties (39), and surface display has been used as an attractive strategy in this context (22,38,47,51).Short metal-binding peptides, such as hexahistidyl peptides, have been introduced into bacterial surface proteins in order to create bacteria with improved metal-binding capacity (21,46). Using this strategy, both Escherichia coli (46) and Staphylococcus carnosus (43) strains with increased ability to bind Ni 2ϩ and Cd 2ϩ ions have been generated. The introduction of combinatorial protein engineering has also made it possible to select peptides, from large peptide libraries with increased selectivity for certain metals (4,30,37,44), and such peptides might become interesting for surface display applications (44).Gram-positive surface display systems have been suggested to exhibit some advantages compared to gram-negative bacteria (28, 49): (i) the translocation involves only a single membrane, and (ii) gram-positive bacteria have been shown to be more rigid and thus less sensitive to shear forces (19, 36) due to the thick cell wall surrounding the cells, and thus potentially more suitable for field applications such as bioadsorption. For metal adsorption applications, gram-positive bacteria have the additional advantage of having inherent metal-binding capacity due to the thick pe...

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Section: Resultssupporting

confidence: 77%

Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Wernérus

Lehtiö

Teeri

et al. 2001

Appl Environ Microbiol

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Multiple affinity domains for the detection, purification and immobilization of recombinant proteins

et al. 1996

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Affinity systems based on specific molecular recognition are valuable tools for detection, purification and immobilization of recombinant proteins. Here, novel multipartite affinity fusion vectors were assembled and investigated to allow flexible binding and elution conditions. The rationale for the assembly of different combinations of affinity domains was to take advantage of the wide variety of molecular interactions of these domains for purification, solubilization, detection and immobilization. In total, seven different affinity tags representing five different types of tag-ligand interactions were studied: (i) monoclonal antibodies-peptides (T7-tag and FLAG peptide); (ii) streptavidin-peptide (Strep-tag); (iii) hexahistidyl-metal ions (His6-tag; (iv) bacterial receptors-serum proteins (staphyloccal protein A-Fc and streptococcal protein G-serum albumin); (v) streptavidin-biotin (in vivo biotinylated peptide). Selected tags were evaluated for the production and purification of Escherichia coli DNA polymerase I (Klenow fragment). On the basis of the results, a vector (pAff2c) was assembled using a novel combination of affinity domains: (i) an in vivo biotinylated peptide; (ii) a His6 sequence, and (iii) a highly soluble serum albumin binding region. Using these three affinities, a wide variety of conditions can be employed for both the binding and the elution steps.

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