Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display

Kronqvist, Nina; Löfblom, John; Severa, Denise; Ståhl, Stefan; Wernérus, Henrik

doi:10.1111/j.1574-6968.2007.00990.x

Cited by 11 publications

(8 citation statements)

References 30 publications

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“…The PCR-amplified PsrP constructs and the gene-synthesized (Eurofins Genomics, Germany) DUF1542 repeat domain of SasC (Uniprot ID: C7BUR8) were cloned into the staphylococcal display vector ‘pHis3C’ using a sequence and ligation independent cloning (SLIC) method5152. Coding sequences of the protein-display constructs were confirmed by DNA sequencing and are listed in the supplemental information.…”

Section: Methodsmentioning

confidence: 99%

“…Bacteria were fixed in 4% paraformaldehyde, washed, and visualized using a fluorescence microscope (Leica Leitz DMRBE). For dotblot assays, surface-displayed proteins were cleaved as previously described52 and applied on nitrocellulose filter membranes. Proteins were detected using polyclonal rabbit-antisera against BR 187–385 , BR 143–156 as well as HRP-coupled anti-His antibodies (ab1187; Abcam, UK).…”

Section: Methodsmentioning

confidence: 99%

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The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

Schulte

Mikaelsson

Beaussart

et al. 2016

Sci Rep

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The major human pathogen Streptococcus pneumoniae is a leading cause of disease and death worldwide. Pneumococcal biofilm formation within the nasopharynx leads to long-term colonization and persistence within the host. We have previously demonstrated that the capsular surface-associated pneumococcal serine rich repeat protein (PsrP), key factor for biofilm formation, binds to keratin-10 (KRT10) through its microbial surface component recognizing adhesive matrix molecule (MSCRAMM)-related globular binding region domain (BR187–385). Here, we show that BR187–385 also binds to DNA, as demonstrated by electrophoretic mobility shift assays and size exclusion chromatography. Further, heterologous expression of BR187–378 or the longer BR120–378 construct on the surface of a Gram-positive model host bacterium resulted in the formation of cellular aggregates that was significantly enhanced in the presence of DNA. Crystal structure analyses revealed the formation of BR187–385 homo-dimers via an intermolecular β-sheet, resulting in a positively charged concave surface, shaped to accommodate the acidic helical DNA structure. Furthermore, small angle X-ray scattering and circular dichroism studies indicate that the aggregate-enhancing N-terminal region of BR120–166 adopts an extended, non-globular structure. Altogether, our results suggest that PsrP adheres to extracellular DNA in the biofilm matrix and thus promotes pneumococcal biofilm formation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

Schulte

Mikaelsson

Beaussart

et al. 2016

Sci Rep

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“…In addition, the vector system was later modified with a 3C protease substrate sequence (Fig. 3 ), which enabled specific proteolytic release of displayed proteins to allow detailed characterization (Kronqvist et al 2008b ).…”

Section: A Surface Display System For S Carnosusmentioning

confidence: 99%

Staphylococcus carnosus: from starter culture to protein engineering platform

Löfblom

Rosenstein

Nguyen

et al. 2017

Appl Microbiol Biotechnol

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Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and “food grade” species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.

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“…The average surface expression level has been estimated in two different studies to around 10 000–100 000 recombinant proteins per cell 14, 79.…”

Section: Gram‐positive Staphylococcal Displaymentioning

confidence: 99%

Bacterial display in combinatorial protein engineering

Löfblom

2011

Biotechnology Journal

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Technologies for display of recombinant protein libraries are today essential tools in many research-intensive fields, such as in the drug discovery processes of biopharmaceutical development. Phage display is still the most widely used method, but alternative systems are available and are becoming increasingly popular. The most rapidly expanding of the alternative systems are the cell display-based technologies, offering innovative strategies for selection and characterization of affinity proteins. Most investigations have focused on eukaryotic yeast for display of protein libraries, but similar systems are also being developed using prokaryotic hosts. This review summarizes the field of bacterial surface display with a strong emphasis on library applications for generation of new affinity proteins. The main focus will be on the most recent progress of the work on primarily Escherichia coli, but also on studies using a recently developed system for display on Gram-positive Staphylococcus carnosus. In addition, general strategies for combinatorial protein engineering using cell display are discussed along with the latest developments of new methodologies with comparisons to mainly phage display technology.

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Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display

Cited by 11 publications

References 30 publications

The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

Staphylococcus carnosus: from starter culture to protein engineering platform

Bacterial display in combinatorial protein engineering

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