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High-affinity bindi...
High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule
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Lindborg, M. (författare)
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- Dubnovitsky, Anatoly (författare)
- Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för molekylärbiologi,Department of Molecular Biology,Karolinska Institute
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- Olesen, Kenneth, 1966 (författare)
- Gothenburg University,Göteborgs universitet,Institutionen för biomedicin,Institute of Biomedicine
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Bjorkman, T. (författare)
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Abrahmsen, L. (författare)
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Feldwisch, J. (författare)
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- Härd, Torleif, 1959 (författare)
- Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för molekylärbiologi,Department of Molecular Biology
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(creator_code:org_t)
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- 2013-08-07
- 2013
- Engelska.
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 26:10, s. 635-644
- Relaterad länk:
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https://academic.oup...
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https://gup.ub.gu.se...
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https://doi.org/10.1...
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https://res.slu.se/i...
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Abstract
Ämnesord
Stäng
- Affibody molecules are engineered binding proteins, in which the three-helix bundle motif of the Z domain derived from protein A is used as a scaffold for sequence variation. We used phage display to select Affibody binders to staphylococcal protein A itself. The best binder, called ZpA963, binds with similar affinity and kinetics to the five homologous E, D, A, B and C domains of protein A, and to a five-domain protein A construct with an average dissociation constant, K-D, of 20 nM. The structure of ZpA963 in complex with the Z domain shows that it interacts with a surface on Z that is identical in the five protein A domains, which explains the multi-domain affinity. This property allows for high-affinity binding by dimeric Affibody molecules that simultaneously engage two protein A domains in a complex. We studied two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively. The dimers both bind protein A with high affinity, very slow off-rates and with saturation-dependent kinetics that can be understood in terms of dimer binding to multiple sites. The head-to-tail (ZpA963)(2)htt dimer binds with an off-rate of k(off) 5 10(6) s(1) and an estimated K-D 16 pM. The results illustrate how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.
Ämnesord
- MEDICIN OCH HÄLSOVETENSKAP -- Klinisk medicin (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Clinical Medicine (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik -- Biokatalys och enzymteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology -- Biocatalysis and Enzyme Technology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- NATURVETENSKAP -- Kemi -- Fysikalisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Physical Chemistry (hsv//eng)
Nyckelord
- molecular recognition
- phage display
- protein engineering
- proteinprotein interactions
- protein
- BACTERIAL RECEPTOR DOMAIN
- STRUCTURAL BASIS
- COMPLEX
- STABILIZATION
- RECOGNITION
- LIBRARIES
- LAGLIO F
- 1995
- JOURNAL OF BIOMOLECULAR NMR
- V6
- P277
- UDIER FW
- 1990
- METHODS IN ENZYMOLOGY
- V185
- P60
- ATES OF AMERICA
- V107
- P15039
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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