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Towards an antibiotic resistance-based assay for protease characterization and engineering
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-9504-4054
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
(English)Manuscript (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.

Keyword [en]
proteases, flow cytometry, CAT, protein engineering, ssrA, ClpXP, tobacco etch virus, TEV, site-specific, high-throughput, selection, proteolysis, libraries
National Category
Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-33548OAI: oai:DiVA.org:kth-33548DiVA: diva2:416060
Note
QC 20110513Available from: 2011-05-13 Created: 2011-05-10 Last updated: 2011-05-16Bibliographically approved
In thesis
1. Intracellular systems for characterization and engineering of proteases and their substrates
Open this publication in new window or tab >>Intracellular systems for characterization and engineering of proteases and their substrates
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Over the years, the view on proteases as relatively non-specific protein degradation enzymes, mainly involved in food digestion and intracellular protein turnover, has shifted and they are now recognized as key regulators of many biological processes that determine the fate of a cell. Besides their biological role, proteases have emerged as important tools in various biotechnical, industrial and medical applications. At present, there are worldwide efforts made that aim at deciphering the biological role of proteases and understanding their mechanism of action in greater detail. In addition, with the growing demand of novel protease variants adapted to specific applications, protease engineering is attracting a lot of attention.

With the vision of contributing to the field of protein science, we have developed a platform for the identification of site-specific proteolysis, consisting of two intracellular genetic assays; one fluorescence-based (Paper I) and one antibiotic resistance-based (Paper IV). More specifically, the assays take advantage of genetically encoded short-lived reporter substrates that upon cleavage by a coexpressed protease confer either increased whole-cell fluorescence or antibiotic resistance to the cells in proportion to the efficiency with which the substrates are processed. Thus, the fluorescence-based assay is highly suitable for high-throughput analysis of substrate processing efficiency by flow cytometry analysis and cell sorting, while the antibiotic resistance assay can be used to monitor and identify proteolysis through (competitive) growth in selective media.

By using the highly sequence specific tobacco etch virus protease (TEVp) as a model in our systems, we could show that both allowed for (i) discrimination among closely related substrate peptides (Paper I & IV) and (ii) enrichment and identification of the best performing substrate-protease combination from a background of suboptimal variants (Paper I & IV). In addition, the fluorescence-based assay was used successfully to determine the substrate specificity of TEVp by flow cytometric screening of large combinatorial substrate libraries (Paper II), and in a separate study also used as one of several methods for the characterization of different TEVp mutants engineered for improved solubility (Paper III).

We believe that our assays present a new and promising path forward for high-throughput substrate profiling of proteases, directed evolution of proteases and identification of protease inhibitors, which all are areas of great biological, biotechnical and medical interest.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. x, 52 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:11
Keyword
proteases, flow cytometry, GFP, CAT, protein engineering, ssrA, ClpXP, tobacco etch virus, TEV, site-specific, high-throughput, selection, proteolysis, libraries
National Category
Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-33549 (URN)978-91-7415-992-9 (ISBN)
Public defence
2011-05-31, Lecture hall FD5 (Svedbergsalen) AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 621-2004-4647
Note
QC 20110516Available from: 2011-05-16 Created: 2011-05-10 Last updated: 2011-05-16Bibliographically approved

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