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Coupled release and site-specific conjugation of Affibody molecules from the surface of E. coli using Sortase A
KTH, School of Biotechnology (BIO), Protein Technology. (Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.)
KTH, School of Biotechnology (BIO), Protein Technology. (ivision of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.)
KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.)ORCID iD: 0000-0001-9423-0541
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Combinatorial protein engineering using libraries displayed on various microorganisms is a powerful method forgeneration of new affinity proteins. Successful efforts often result in broad panels of isolated binders, which are thentypically subcloned, produced, purified and characterized in various assays. Many such assays also require conjugation tofor example reporters or other functional molecules and the downstream production and modification thus tends to be verylaborious and limits the number of candidates that can be screened. Staphylococcal sortase A is a natural transpeptidasethat catalyzes the ligation between a LPXTG motif and N-terminal glycines and is today used in a variety of applicationsfor site-specific conjugation of different molecules to recombinant proteins. We have previously developed a surfacedisplay method for combinatorial protein engineering of Affibody molecules on the outer membrane of E. coli usingautodisplay. Here, we introduced a sortase-A recognition motif into the displayed recombinant proteins and evaluatedsortase-mediated release and specific conjugation of various reporters to Affibody molecules. The approach has potentialto significantly increase the flexibility and throughput of downstream characterization of affinity proteins after directedevolution using cell display and FACS.

Keywords [en]
AIDA-I, Affibody molecules, autodisplay, autotransporter, bioconjugation, combinatorial protein engineering, directed evolution, protein ligation, sortase A
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-213450OAI: oai:DiVA.org:kth-213450DiVA, id: diva2:1137348
Note

QC 20170904

Available from: 2017-08-31 Created: 2017-08-31 Last updated: 2017-09-04Bibliographically approved
In thesis
1. Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging
Open this publication in new window or tab >>Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Directed evolution is today an established strategy for generation of new affinity proteins. This thesis describes the development of a cell-display method using Escherichia coli for directed evolution of Affibody molecules. Further, the thesis describes rational design of Affibody-based tracers, intended for future patient stratification using medical imaging. Fusing recombinant proteins to various autotransporters is a promising approach for efficient surface display on the surface of E. coli, as well as for construction of high-complexity libraries. In paper I, we successfully engineered an expression vector for display of Affibody molecules using the autotransporter AIDA-I. In paper II, a large Affibody library of 2.3x109 variants was constructed and screening using FACS resulted in new specific binders in the nanomolar range. In paper III, we demonstrated Sortase-mediated secretion and conjugation of binders directly from the E. coli surface. 

The three following studies describe rational design of Affibody-based tracers against two cancer-associated targets for molecular imaging. First, anti-HER3 Affibody molecules were labelled with 111In, and SPECT imaging showed that the conjugates specifically targeted HER3-expressing xenografts. Furthermore, labeling with 68Ga for PET imaging showed that tumor uptake correlated with HER3 expression, suggesting that the tracers have potential for patient stratification. The last study describes the development and investigation of anti-EGFR Affibody-based imaging agents. Labeled with 89Zr, the Affibody tracer demonstrated higher tumor uptake at 3 h post injection than the anti-EGFR antibody cetuximab at 48 h post injection. 

In conclusion, this thesis describes new tools and knowledge that will hopefully contribute to the development of affinity proteins for biotechnology, therapy and medical imaging in the future. 

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. p. 80
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:17
Keywords
directed evolution, microbial display, E. coli, Affibody molecule, autotransporter, medical imaging, HER receptor family, Sortase A
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-213451 (URN)978-91-7729-504-4 (ISBN)
Public defence
2017-10-06, F3, Lindstedtsvägen 26, Sing-Sing, floor 2, KTH Campus, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20170904

Available from: 2017-09-05 Created: 2017-08-31 Last updated: 2017-09-05Bibliographically approved

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Andersson, Ken G.Löfblom, John

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