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  • 1. Cano, F.
    et al.
    Plotnicky-Gilquin, H.
    Nguyen, T. N.
    Liljeqvist, S.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Bonnefoy, J. Y.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Robert, A.
    Partial protection to respiratory syncytial virus (RSV) elicited in mice by intranasal immunization using live staphylococci with surface-displayed RSV-peptides2000In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 18, no 24, p. 2743-2752Article in journal (Refereed)
    Abstract [en]

    A live bacterial vaccine-delivery system based on the food-grade bacterium Staphylococcus carnosus was used for delivery of peptides from the G glycoprotein of human respiratory syncytial virus, subtype A (RSV-A). Three peptides, corresponding to the G protein amino acids, 144-159 (denoted G5), 190-203 (G9) and 171-188 (G4 S), the latter with four cysteine residues substituted for serines, were expressed by recombinant means as surface-exposed on three different bacteria, and their surface accessibility on the bacteria was verified by fluorescence-activated cell sorting (FACS). Intranasal immunization of mice with the live recombinant staphylococci elicited significant anti-peptide as well as anti-virus serum IgG responses of balanced IgG1/IgG2a isotype profiles, and upon viral challenge with 10(5) tissue culture infectious doses(50) (TCID50), lung protection was demonstrated for approximately half of the mice in the G9 and G4 S immunization groups. To our knowledge, this is the first study in which protective immunity to a viral pathogen has been evoked using food-grade bacteria as vaccine-delivery vehicles.

  • 2.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Do, T. -H
    Lüthje, P.
    Tran, N. T.
    Brauner, A.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Truong, N. H.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, article id 47Article in journal (Refereed)
    Abstract [en]

    Background: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the β-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. Results: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. Conclusions: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.

  • 3. Hansson, M.
    et al.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Gunneriusson, E.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Surface display on gram positive bacteria2001In: Combinatorial chemistry & high throughput screening, ISSN 1386-2073, E-ISSN 1875-5402, Vol. 4, no 2, p. 171-184Article in journal (Refereed)
    Abstract [en]

    Heterologous surface display on Gram-positive bacteria was first described almost a decade ago and has since then developed into an active research area. Gram-positive bacterial surface display has today found a range of applications, in immunology, microbiology and biotechnology. Live bacterial vaccine delivery vehicles are being developed through the surface display of selected foreign antigens on the bacterial surfaces. In this field, second generation vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered Gram-positive bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other variants of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. This article explains the basis of Grampositive bacterial surface display, and discusses current uses and possible future trends of this emerging technology.

  • 4. Hansson, M.
    et al.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Nguyen, T. N.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae2002In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 210, no 2, p. 263-270Article in journal (Refereed)
    Abstract [en]

    General expression vectors, designed for intracellular expression or secretion of recombinant proteins in the non-pathogenic Staphylococcus carnosus, were constructed. Both vector systems encode two different affinity tags, an upstream albumin binding protein and a downstream hexahistidyl peptide, and are furnished with cleavage sites for two site-specific proteases for optional affinity tag removal. To evaluate the novel vectors, the gene encoding the outer membrane protein A (OmpA) of Klebsiella pneumoniae was introduced into the vectors. Efficient production was demonstrated in both systems, although, as expected for OmpA fusions, somewhat better intracellularly, and the fusion proteins could be recovered as full-length products by affinity chromatography.

  • 5.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Do, Thi-Huyen
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli2012In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 11, article id 118Article in journal (Refereed)
    Abstract [en]

    Background: The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H: gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results: The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His(6)-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H: gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His(6)-tag facing the medium. Conclusions: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His(6)-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

  • 6.
    Kostallas, George
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Löfdahl, Per-Ake
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay2011In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 1, p. e16136-Article in journal (Refereed)
    Abstract [en]

    Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P19, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates.

  • 7.
    Kostallas, George
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates2010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 22, p. 7500-7508Article in journal (Refereed)
    Abstract [en]

    We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1: 100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.

  • 8.
    Kostallas, George
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sandersjöö, Lisa
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Al-Askeri, Mohammed
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Construction, expression and characterization of TEV protease mutants engineered for improved solubilityManuscript (preprint) (Other academic)
    Abstract [en]

    In recent years, the highly sequence specific tobacco etch virus protease (TEVp) has emerged as one of the most popular and widely used reagents for removal of fusion tags from target proteins. Its use, however, has been hampered due to relatively poor solubility and inefficient expression in E. coli. Although a lot of progress has been made, there is still need for new and improved TEVp variants. Recently, two different gain-of-function TEVp mutants were described; one containing the substitutions L56V/S135G, which conferred improved solubility and activity in vitro, while the other mutant, containing the substitutions T17S/N68D/I77V, was claimed to yield more soluble protease than the wild-type (wt) protease upon overexpression in E. coli. Here, we analyzed if the L56V/S135G substitutions could promote increased solubility also in vivo, as that would be beneficial to TEVp production and had never been investigated before. We also intended to create a novel, and hopefully superior, TEVp variant with all five mutations combined (T17S/L56V/N68D/S135G/I77V) in a single protease molecule. This variant and the two parental TEVp variants as well as the wt protease, were all expressed in E. coli and characterized with respect to the expression levels, solubility and activity using several different techniques; among them, a newly developed fluorescence-assisted whole-cell assay that directly reports on the apparent protease activity in vivo. Our results show that the L56V/S135G substitutions improve the solubility not only in vitro but also in vivo, which did hold true for the activity as well. Disappointingly, the protease variant containing all five substitutions (T17S/L56V/N68D/S135G/I77V) did not show the best performance, which instead the L56V/S135G variant did. In contrast to an earlier report, we show that the substitutions T17S/N68D/I77V, did not improve the TEVp solubility. In fact, they reduced the activity, and even appeared to have a slightly negative effect on solubility, of all protease constructs in which they were present. Thus, the best current and most promising TEVp variant for future protease engineering efforts, towards improved expression properties and enhanced catalytic efficiency, are those containing the L56V/S135G substitutions.

  • 9. Lehtio, J.
    et al.
    Wernérus, Henrik
    KTH, Superseded Departments, Biotechnology.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain2001In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 195, no 2, p. 197-204Article in journal (Refereed)
    Abstract [en]

    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 Tor the generation of whole-cell microbial tools Fur different applications will be discussed.

  • 10. Nguyen, N
    et al.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Merle-Poitte, C
    Robert, A
    Baussant, T
    Haeuw, F
    Uhlen, Mathias
    KTH, Superseded Departments, Biotechnology.
    Binz, H
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Chromosomal sequencing using a PCR-based biotin-capture method allowed isolation of the complete gene for the outer membrane protein A of Klebsiella pneumoniae1998In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 210, no 1, p. 93-101Article in journal (Refereed)
    Abstract [en]

    By employing a novel biotin-and PCR-assisted capture method, which allows determination of unknown sequences on chromosomal DNA. the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate fragments with only partly determined nt sequences. The homology of the sequenced gene to ompAs of related bacteria is discussed. The ompA gene was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed. (C) 1998 Elsevier Science B.V.

  • 11.
    Samuelson, Patrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gunneriusson, E.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Display of proteins on bacteria2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 2, p. 129-154Article, review/survey (Refereed)
    Abstract [en]

    Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and disc-asses current uses and possible future trends of these emerging technologies.

  • 12.
    Samuelson, Patrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wernérus, Henrik
    KTH, Superseded Departments, Biotechnology.
    Svedberg, M.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Staphylococcal surface display of metal-binding polyhistidyl peptides2000In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 66, no 3, p. 1243-1248Article in journal (Refereed)
    Abstract [en]

    Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni2+- and Cd2+-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.

  • 13.
    Sandersjöö, Lisa
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Kostallas, George
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 1, p. 155-162Article in journal (Refereed)
    Abstract [en]

    Proteases are involved in many biological processes and have become important tools in biomedical research and industry. Technologies for engineering and characterization of, for example, proteolytic activity and specificity are essential in protease research. Here, we present a novel method for assessment of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that, upon processing by a co-expressed protease, confer antibiotic resistance to bacteria in proportion to the cleavage efficiency. We have demonstrated that cells co-expressing cleavable reporters together with tobacco etch virus protease (TEVp) could be discriminated from cells with non-cleavable reporters by growth in selective media. Importantly, the resistance to antibiotics proved to correlate with the substrate processing efficiency. Thus, by applying competitive growth of a mock library in antibiotic-containing medium, we could show that the substrate preferred by TEVp was enriched relative to less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases and protease recognition sequences towards improved or even new functionality.

  • 14.
    Sandersjöö, Lisa
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Kostallas, George
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Towards an antibiotic resistance-based assay for protease characterization and engineeringManuscript (preprint) (Other academic)
    Abstract [en]

    Proteases attract a lot of interest, not only because of their involvement in many biological processes, but also as essential tools in biomedical research and industry. Here, we present a novel genetic method for identification of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that upon processing by a coexpressed protease confer antibiotic resistance to cells in proportion to the cleavage efficiency.

    We demonstrate that cells expressing cleavable or non-cleavable reporters together with tobacco etch virus protease (TEVp), could be distinguished from each other by growth in selective media. Moreover, the growth rate proved to correlate with the substrate processing efficiency. Thus by applying competitive growth in antibiotic-containing medium, we could also show that the substrate preferred by TEVp was enriched at the expense of other less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases towards improved and/or new functionality.

  • 15. Ståhl, Stefan
    et al.
    Robert, A.
    Gunneriusson, E.
    Wernérus, Henrik
    KTH, Superseded Departments, Biotechnology.
    Cano, F.
    Liljeqvist, S.
    Hansson, M.
    Nguyen, T. N.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Staphylococcal surface display and its applications2000In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 290, no 7, p. 571-577Article, review/survey (Refereed)
    Abstract [en]

    Novel surface proteins can be introduced onto the bacterial cell surface by recombinant means. Here, we describe the development of such display systems for two food-grade bacteria, Staphylococcus carnosus and Staphylococcus xylosus, and present how such engineered bacteria can be used in different applications. A study will be described in which such staphylococci were employed as vaccine delivery vehicles to elicit protective antibody responses to respiratory syncytial virus (RSV). The use of surface-engineered staphylococci as novel microbial biocatalysts, as a new type of whole-cell diagnostic devices or for adsorption of metal ions with potential environmental or biosensor applications, will also be discussed.

  • 16.
    Wernérus, Henrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lehtiö, Janne
    KTH, Superseded Departments, Biotechnology.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Engineering of staphylococcal surfaces for biotechnological applications2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 1, p. 67-78Article, review/survey (Refereed)
    Abstract [en]

    Novel surface proteins can be introduced onto bacterial cell surfaces by recombinant means. Here, we describe various applications of two such display systems for the food-grade bacteria Staphylococcus carnosus and Staphylococcus xylosus, respectively. The achievements in the use of such staphylococci as live bacterial vaccine delivery vehicles will be described. Co-display of proteins and peptides with adhesive properties to enable targeting of the bacteria, have significantly improved the vaccine delivery potential. Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization using such 'second generation' vaccine delivery systems. Furthermore, antibody fragments and other 'affinity proteins' with capacity to specifically bind a certain protein, e.g. Staphylococcus aureus protein A-based affibodies, have been surface-displayed on staphylococci as initial efforts to create whole-cell diagnostic devices. Surface display of metal-binding peptides, or protein domains into which metal binding properties has been engineered by combinatorial protein engineering, have been exploited to create staphylococcal bioadsorbents for potential environmental or biosensor applications. The use of these staphylococcal surface display systems as alternatives for display of large protein libraries and subsequent affinity selection of relevant binding proteins by fluorescence-activated cell sorting (FACS) will be discussed.

  • 17.
    Wernérus, Henrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Fluorescence-activated cell sorting of specific affibody-displaying staphylococci2003In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 69, no 9, p. 5328-5335Article in journal (Refereed)
    Abstract [en]

    Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.

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