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Generating a High Valency Biotin Binder by Selecting Uniform Protein Assemblies via Crystallization

1
Department of Biological Chemistry, The Alexander Silverman Institute of Life Sciences, The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, The Edmond J. Safra Campus, Jerusalem 91904, Israel
2
Center for Nanoscience and Nanotechnology, and The Alexander Silverman Institute of Life Sciences, The Hebrew University of Jerusalem, The Edmond J. Safra Campus, Jerusalem 91904, Israel
*
Author to whom correspondence should be addressed.
Crystals 2019, 9(7), 353; https://doi.org/10.3390/cryst9070353
Received: 28 May 2019 / Revised: 3 July 2019 / Accepted: 9 July 2019 / Published: 11 July 2019
(This article belongs to the Special Issue X-ray Crystallography)
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Abstract

Crystallization is a common practice in the purification process in small molecule synthesis while selecting the wanted product. For proteins it is rarely applied due to the methodological predicaments in obtaining crystals. Our observation of the stabilized octamers in the crystal structure of hoefavidin, a novel dimeric member of the avidin family, led to the notion of developing a novel biotechnological tool via covalent crosslinking. The avidin–biotin system has been exploited for decades utilizing the ultra-high affinity between avidin and biotin as a basis for numerous applications. Optimizing the system led to the discovery of a novel group of dimeric avidins including hoefavidin. Hoefavidin has a dynamic quaternary structure, where a dimer is the basis for generating the octamer via crystallographic symmetry operation. Upon biotin binding in solution hoefavidin dissociates solely into dimers. In order to stabilize the octamer, we designed the P61C mutant to form a disulfide bridge stabilizing the octamer and preventing dissociation upon biotin binding. The process of selecting P61C hoefavidin uniform octamers includes crystallization followed by dissolving the crystals. The P61C modified hoefavidin octamer can have a substantial added value to the various biotechnological applications and advances of the biotin based high affinity systems. View Full-Text
Keywords: protein crystallography; crystal packing; avidin; biotin; nanotechnology protein crystallography; crystal packing; avidin; biotin; nanotechnology
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Avraham, O.; Levi-Kalisman, Y.; Livnah, O. Generating a High Valency Biotin Binder by Selecting Uniform Protein Assemblies via Crystallization. Crystals 2019, 9, 353.

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