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Bacteria-based methods for engineering and characterization of proteases and affinity proteins
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0001-9504-4054
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: urn:nbn:se:kth:diva-166645ISBN: 978-91-7595-525-4 (print)OAI: oai:DiVA.org:kth-166645DiVA: diva2:811681
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
List of papers
1. A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance
Open this publication in new window or tab >>A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance
2014 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, 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.

Keyword
Protease engineering, Protease substrate profiling, Selection system, Tobacco Etch Virus
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-140898 (URN)10.1002/biot.201300234 (DOI)000337540100016 ()2-s2.0-84891908926 (Scopus ID)
Funder
Swedish Research Council, 2012-5236 2004-4667
Note

QC 20140204

Available from: 2014-02-04 Created: 2014-02-04 Last updated: 2017-12-06Bibliographically approved
2. A new prodrug form of Affibody molecules (pro-Affibody) is selectively activated by cancer-associated proteases
Open this publication in new window or tab >>A new prodrug form of Affibody molecules (pro-Affibody) is selectively activated by cancer-associated proteases
2015 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 72, no 7, 1405-1415 p.Article in journal (Refereed) Published
Abstract [en]

Affinity proteins have advanced the field of targeted therapeutics due to their generally higher specificity compared to small molecular compounds. However, side effects caused by on-target binding in healthy tissues are still an issue. Here, we design and investigate a prodrug strategy for improving tissue specificity of Affibody molecules in future in vivo studies. The prodrug Affibody (pro-Affibody) against the HER2 receptor was constructed by fusing a HER2-specific Affibody (ZHER2) to an anti-idiotypic Affibody (anti-ZHER2). The linker was engineered to comprise a substrate peptide for the cancer-associated matrix metalloprotease 1 (MMP-1). The hypothesis was that the binding surface of ZHER2 would thereby be blocked from interacting with HER2 until the substrate peptide was specifically hydrolyzed by MMP-1. Binding should thereby only occur where MMP-1 is overexpressed, potentially decreasing on-target toxicities in normal tissues. The pro-Affibody was engineered to find a suitable linker and substrate peptide, and the different constructs were evaluated with a new bacterial display assay. HER2-binding of the pro-Affibody was efficiently masked and proteolytic activation of the best variant yielded over 1,000-fold increase in apparent binding affinity. Biosensor analysis revealed that blocking of the pro-Affibody primarily affected the association phase. In a cell-binding assay, the activated pro-Affibody targeted native HER2 on cancer cells as opposed to the non-activated pro-Affibody. We believe this prodrug approach with proteolytic activation is promising for improving tissue specificity in future in vivo targeting applications and can hopefully be extended to other Affibody molecules and similar affinity proteins as well.

Keyword
Bacterial display, Cell surface display, Combinatorial protein engineering, Directed evolution, Flow cytometry, HER2, MMP-1, Staphylococcal display
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-160032 (URN)10.1007/s00018-014-1751-8 (DOI)000351455100014 ()25287047 (PubMedID)2-s2.0-84907611403 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150419

Available from: 2015-02-13 Created: 2015-02-13 Last updated: 2017-12-04Bibliographically approved
3. Bacterial display of self-blocking affinity proteins for efficient protease substrate profiling
Open this publication in new window or tab >>Bacterial display of self-blocking affinity proteins for efficient protease substrate profiling
(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
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:nbn:se:kth:diva-166537 (URN)
Note

QS 20150521

Available from: 2015-05-12 Created: 2015-05-11 Last updated: 2015-05-21Bibliographically approved
4. Development of a fluorescence-based intracellular method for function-based isolation of protein-based aggregation inhibitors
Open this publication in new window or tab >>Development of a fluorescence-based intracellular method for function-based isolation of protein-based aggregation inhibitors
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-166644 (URN)
Note

QS 2015

Available from: 2015-05-12 Created: 2015-05-12 Last updated: 2015-09-22Bibliographically approved

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