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Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
2010 (engelsk)Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, nr 22, s. 7500-7508Artikkel i tidsskrift (Fagfellevurdert) Published
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.

sted, utgiver, år, opplag, sider
2010. Vol. 76, nr 22, s. 7500-7508
Emneord [en]
ETCH VIRUS PROTEASE, DIRECTED EVOLUTION, STRUCTURAL GENOMICS, RATIONAL DESIGN, IN-VIVO, SPECIFICITY, DEGRADATION, EXPRESSION, PROTEINS, ENZYMES
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-27102DOI: 10.1128/AEM.01558-10ISI: 000283843900017Scopus ID: 2-s2.0-78649692108OAI: oai:DiVA.org:kth-27102DiVA, id: diva2:375777
Merknad
QC 20101209Tilgjengelig fra: 2010-12-09 Laget: 2010-12-06 Sist oppdatert: 2017-12-11bibliografisk kontrollert
Inngår i avhandling
1. Intracellular systems for characterization and engineering of proteases and their substrates
Åpne denne publikasjonen i ny fane eller vindu >>Intracellular systems for characterization and engineering of proteases and their substrates
2011 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2011. s. x, 52
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2011:11
Emneord
proteases, flow cytometry, GFP, CAT, protein engineering, ssrA, ClpXP, tobacco etch virus, TEV, site-specific, high-throughput, selection, proteolysis, libraries
HSV kategori
Forskningsprogram
SRA - Molekylär biovetenskap
Identifikatorer
urn:nbn:se:kth:diva-33549 (URN)978-91-7415-992-9 (ISBN)
Disputas
2011-05-31, Lecture hall FD5 (Svedbergsalen) AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Swedish Research Council, 621-2004-4647
Merknad
QC 20110516Tilgjengelig fra: 2011-05-16 Laget: 2011-05-10 Sist oppdatert: 2011-05-16bibliografisk kontrollert

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