Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 507423
Title Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis
Author(s) Geuijen, K.P.M.; Egging, David F.; Bartels, S.; Schouten, Jan; Schasfoort, Richard B.; Eppink, M.H.M.
Source Protein Science 25 (2016)10. - ISSN 0961-8368 - p. 1841 - 1852.
Department(s) Bioprocess Engineering
Publication type Refereed Article in a scientific journal
Publication year 2016
Abstract Chemical protein biotinylation and streptavidin or anti-biotin-based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine-tuning of critical parameters is important to reduce biotinylation in a protein binding site.
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