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Bacterial display of self-blocking affinity proteins for efficient protease substrate profiling
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
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Proteases are involved in fundamental biological processes and are important tools in both industry and biomedical research. One of the most important mechanisms of a protease is its ability to discriminate among potential substrates. Here, we present a new method for substrate profiling of proteases. The substrates are displayed within a fusion protein on the surface of the Gram-positive bacteria S. carnosus. The substrate is inserted in a linker, which connects two anti-idiotypic Affibody molecules (ZHER2 and anti-ZHER2). ZHER2 has affinity for the HER2 receptor and anti-ZHER2 binds to ZHER2, thereby blocking the binding surface from interacting with HER2. By site-specific proteolysis of the substrate within the linker, the blocking domain is released and will diffuse away, thus the HER2 binding capacity of ZHER2 is restored. The proteolysis is therefore reflected in HER2 binding, which can be analyzed by flow cytometry upon labeling with fluorescent HER2. By applying this methodology we could enrich for cells displaying a substrate peptide, efficiently hydrolyzed by tobacco etch virus protease (TEVp), from a library of cells displaying different substrates. In an attempt to determine the substrate preference of matrix metalloprotease-1 (MMP-1), cells displaying a previously reported motif (PXXXHy) were enriched. On-cell determination of apparent kcat/KM revealed that the enriched substrate peptides were hydrolyzed 6-8 times more efficiently than a previously reported substrate peptide.

Keyword [en]
Protease, substrate profiling, bacterial display, combinatorial protein engineering, staphylococcal display, directed evolution, cell surface display, flow cytometry, MMP-1, tobacco etch virus protease, TEVp, HER2
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-166537OAI: oai:DiVA.org:kth-166537DiVA: diva2:811677
Note

QS 20150521

Available from: 2015-05-12 Created: 2015-05-11 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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:10
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
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)
Opponent
Supervisors
Note

QC 20150521

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

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Sandersjöö, LisaLöfblom, John

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