Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0001-9504-4054
KTH, School of Biotechnology (BIO), Protein Technology.
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0001-9423-0541
2017 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 12, no 1, 1600365Article in journal (Refereed) Published
Abstract [en]

Proteases are involved in fundamental biological processes and are important tools in both biotechnological and biomedical research. An important property of proteases is to discriminate among potential substrates. Here, a new method for substrate profiling of proteases is presented. The substrates are displayed between two anti-idiotypic affinity domains on the Gram-positive bacterium Staphylococcus carnosus. The first domain functions as a reporter tag and has affinity for a labeled reporter protein, whereas the second domain blocks the reporter tag from interacting with the reporter protein. Site-specific proteolysis of the substrate results in release of the blocking domain, enabling the reporter tag to bind the labeled reporter protein. Proteolysis is therefore reflected in reporter binding, which is quantified by flow cytometry. First, the method with tobacco etch virus protease (TEVp) is evaluated and then the substrate preference of matrix metalloprotease-1 (MMP-1) is determined using two libraries of around three million substrates each. Identified substrate peptides contained the previously reported motif (PXXXHy) and on-cell determination of apparent kcat/KM revealed that the enriched substrate peptides are hydrolyzed six to eight-fold more efficiently than a previously reported substrate peptide. The method thus works as intended and the authors believe it has potential as an efficient tool for substrate profiling. 

Place, publisher, year, edition, pages
2017. Vol. 12, no 1, 1600365
Keyword [en]
Flow cytometry, Matrix Metalloprotease, Protease substrate profiling, Screening method, Tobacco Etch Virus protease, Bacteria, Bins, Peptides, Proteins, Proteolysis, Tobacco, Viruses, Biological process, Biomedical research, Gram-positive bacterium, Potential substrate, Screening methods, Substrates, hybrid protein, interstitial collagenase, peptide hydrolase, peptide library, proteinase, TEV protease, enzyme specificity, enzymology, metabolism, procedures, protein degradation, protein engineering, Staphylococcus, Endopeptidases, Matrix Metalloproteinase 1, Peptide Hydrolases, Recombinant Fusion Proteins, Substrate Specificity
National Category
Medical Engineering
Identifiers
URN: urn:nbn:se:kth:diva-202243DOI: 10.1002/biot.201600365ScopusID: 2-s2.0-85006093230OAI: oai:DiVA.org:kth-202243DiVA: diva2:1082894
Note

Correspondence Address: Löfblom, J.; Division of Protein Technology, School of Biotechnology, KTH – Royal Institute of Technology, AlbaNova University CenterSweden; email: lofblom@kth.se. QC 20170320

Available from: 2017-03-20 Created: 2017-03-20 Last updated: 2017-03-20Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Search in DiVA

By author/editor
Sandersjöö, LisaLöfblom, John
By organisation
Protein Technology
In the same journal
Biotechnology Journal
Medical Engineering

Search outside of DiVA

GoogleGoogle Scholar

Altmetric score

CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf