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A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance
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
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
2014 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 9, no 1, 155-162 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2014. Vol. 9, no 1, 155-162 p.
Keyword [en]
Protease engineering, Protease substrate profiling, Selection system, Tobacco Etch Virus
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-140898DOI: 10.1002/biot.201300234ISI: 000337540100016ScopusID: 2-s2.0-84891908926OAI: diva2:693508
Swedish Research Council, 2012-5236 2004-4667

QC 20140204

Available from: 2014-02-04 Created: 2014-02-04 Last updated: 2015-05-21Bibliographically approved
In thesis
1. Bacteria-based methods for engineering and characterization of proteases and affinity proteins
Open this publication in new window or tab >>Bacteria-based methods for engineering and characterization of proteases and affinity proteins
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is focused on the development of methods for characterization and engineering of both proteases and affinity proteins. In addition, a prodrug concept for small affinity proteins is developed.

Two of the developed methods are for engineering and/or characterization of proteases. First, a method for substrate profiling and engineering of proteases was investigated (paper I). In this method, a protease and a reporter are co-expressed in E. coli. The reporter is comprised of an enzyme, which confers resistance to an antibiotic, fused to a substrate, and a degradation tag. In absence of site-specific proteolysis within the reporter, the degradation tag renders the entire reporter a substrate for the intracellular degradation machinery. Thus, by applying competitive growth in presence of the antibiotic, a substrate that is preferred by a model protease could be enriched relative to less efficiently hydrolyzed substrates. Then, an alternative method for substrate profiling was developed (paper III). Here, the substrate is instead displayed on the surface of bacteria, and located between two anti-idiotypic domains, where one blocks the other from interacting with a reporter. Site-specific proteolysis releases the blocking domain and is therefore reflected in reporter binding. After incubation with fluorescently labeled reporter, the proteolysis can be analyzed by flow cytometry. When large libraries of potential substrates for matrix metalloprotease 1 (MMP-1) were screened, a panel of substrates with the previously reported motif PXXXHy was enriched, thereby demonstrating the potential of the method. This method offers the possibility for high-throughput substrate profiling of proteases as well as engineering of substrates for use in for example protease-activated prodrugs.

In another study, a new prodrug concept for small affinity proteins was developed to improve the tissue selectivity in future in vivo studies (paper II). This concept takes advantage of the local upregulation of proteases in the diseased tissue in various disorders. By fusing a targeting domain to an anti-idiotypic binding partner via a protease-sensitive linker, the targeting domain is masked from interacting with its target until activation by site-specific proteolysis within the linker. The concept was demonstrated for a small affinity protein (Affibody molecule). Bacterial display was employed to engineer the so-called pro-Affibody. When displayed on the bacterial surface, the pro-Affibody showed over 1.000-fold increase in apparent binding affinity upon activation by a disease-associated protease. Additionally, the activated pro-Affibody could bind to its target expressed on cancer cells, as opposed to the non-activated pro-Affibody. This concept is likely to be extendable to other small affinity proteins and opens up for the possibility to develop new such prodrugs to previously non-druggable targets.

In the last study, a screening method for protein-based aggregation inhibitors was developed (paper IV). In this method, a reporter and an inhibitor are co-expressed in E. coli. The reporter is comprised of green fluorescent protein (GFP) fused to an aggregation prone peptide. Upon aggregation, the fluorescence is decreased, but it is then restored when the reporter is co-expressed with an inhibitor. In a model screening experiment, an Affibody molecule that targets the Aβ peptide (involved in Alzheimer’s disease) could be enriched from a background of non-inhibiting Affibody molecules. Also this method is likely to be extendable to other types of affinity proteins, and also to different aggregation prone peptides/proteins involved in other diseases.

In conclusion, the methods and concepts presented in this thesis could in the future yield new means for the engineering and characterization of proteins with desired properties to be used in both biotechnological and medical applications.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. 58 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:10
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-166645 (URN)978-91-7595-525-4 (ISBN)
Public defence
2015-06-05, FR4 AlbaNova Universitetscentrum, KTH, Roslagstullsbacken 21, Stockholm, 13:00 (English)

QC 20150521

Available from: 2015-05-21 Created: 2015-05-12 Last updated: 2015-05-21Bibliographically approved

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Sandersjöö, LisaKostallas, GeorgeLöfblom, JohnSamuelson, Patrik
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