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Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coli
KTH, Skolan för bioteknologi (BIO), Proteinteknologi. (Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.)
KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden.
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0002-9282-0174
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0001-9423-0541
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Abstract [en]

Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is the most frequently used method, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli have several properties that are valuable for library applications and then in particular the high transformation efficiency. Although the first studies on display of recombinant peptides and proteins on E. coli were reported over 25 years ago, the method is still not fully established for directed evolution of affinity proteins. More recently, the use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and in particular for directed evolution of different enzymes. Here, we report on display of a large naïve Affibody library on the outer membrane of E. coli using the autotransporter AIDA-I. The expression cassette was first engineered by removing non-essential sequences, followed by introduction of an Affibody library, comprising more than 109 variants, into the new display vector. Selections by FACS against five different target molecules resulted in a panel of binders with down to nanomolar affinities.

Emneord [en]
Bacterial display, affibody library, AIDA-I, autodisplay, autotransporter, directed evolution, FACS
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-213448OAI: oai:DiVA.org:kth-213448DiVA, id: diva2:1137344
Merknad

QC 20170904

Tilgjengelig fra: 2017-08-31 Laget: 2017-08-31 Sist oppdatert: 2017-12-18bibliografisk kontrollert
Inngår i avhandling
1. Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging
Åpne denne publikasjonen i ny fane eller vindu >>Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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. 

 

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2017. s. 80
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:17
Emneord
directed evolution, microbial display, E. coli, Affibody molecule, autotransporter, medical imaging, HER receptor family, Sortase A
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-213451 (URN)978-91-7729-504-4 (ISBN)
Disputas
2017-10-06, F3, Lindstedtsvägen 26, Sing-Sing, floor 2, KTH Campus, Stockholm, 13:00 (engelsk)
Opponent
Veileder
Merknad

QC 20170904

Tilgjengelig fra: 2017-09-05 Laget: 2017-08-31 Sist oppdatert: 2017-09-05bibliografisk kontrollert

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