WFRF:(Andersson Ken G. G.)
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Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coli
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- Andersson, Ken G. (författare)
- KTH,Proteinteknologi,Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
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- Persson, Jonas (författare)
- KTH,Proteinteknologi,Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden
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- Ståhl, Stefan (författare)
- KTH,Proteinteknologi
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- Engelska.
- Annan publikation (övrigt vetenskapligt/konstnärligt)
Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Nyckelord
- Bacterial display
- affibody library
- AIDA-I
- autodisplay
- autotransporter
- directed evolution
- FACS
- Bioteknologi
- Biotechnology
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- Andersson, Ken G ...
- Persson, Jonas
- Ståhl, Stefan
- Löfblom, John
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- NATURVETENSKAP
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