Geuijen Karin P M, Egging David F, Bartels Stefanie, Schouten Jan, Schasfoort Richard B, Eppink Michel H
Downstream Processing, Synthon Biopharmaceuticals, BV, 6503, GN Nijmegen, The Netherlands.
Bioprocess Engineering, Wageningen University, 6700 AA, Wageningen, The Netherlands.
Protein Sci. 2016 Oct;25(10):1841-52. doi: 10.1002/pro.2994. Epub 2016 Aug 19.
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.
化学蛋白质生物素化以及基于链霉亲和素或抗生物素的捕获方法常用于蛋白质研究，这是一种比将蛋白质直接偶联到生物传感器表面更可控的替代方法。生物素化后，目标相互作用位点可能会被生物素基团阻断，从而降低蛋白质的表观活性。最小化生物素化可以避免表观活性的损失，但在标记参与结合的氨基酸时，目标结合位点仍可能被阻断。在此，我们描述了最小化标记的反应条件优化研究。我们选择低亲和力Fcγ受体作为模型化合物，因为这些蛋白质在其活性结合位点含有许多赖氨酸，因此为最小化标记方法提供了一个有趣的系统。我们能够确定最小化标记方法中最关键的参数（蛋白质与生物素的比例和孵育pH值），在该方法中，所选蛋白质对分析物结合仍保持最高活性。通过质谱肽图谱对最小化标记材料上生物素化的定位与结合试验中的蛋白质活性相关。我们表明，仅追求最小化标记不足以维持蛋白质的活性。仔细微调关键参数对于减少蛋白质结合位点的生物素化很重要。
