Govender S, Jacobs E P, Bredenkamp M W, Swart P
Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Nov 1;859(1):1-8. doi: 10.1016/j.jchromb.2007.08.025. Epub 2007 Aug 26.
A novel, reusable biotinylated affinity chromatography strategy for the bio-specific binding of bioactive avidin tagged enzymes or polypeptides is reported. Using an avidin coupled peroxidase fusion protein as a test system; non-specific protein shielding and matrix regeneration were also shown. The amphiphilic surfactant Pluronic F108 was used as an affinity linker, by non-covalent binding to membrane chromatographic matrices while the terminal hydroxyl groups of Pluronic were covalently coupled to the biological ligand biotin. Planar nonporous membranes of varying surface chemistry were synthesised to test the matrix dependent affinity binding of biotinylated Pluronic and their respective ability to resist non-specific protein adsorption. Membrane regeneration using sodium dodecyl sulphate (SDS) was capable of displacing both adsorbed proteins and Pluronic. SDS micelles (34 mM) were effective in desorbing membrane bound protein while 5mM SDS removed up to 85% of the bound ligand after 20 h incubation at 20 degrees C. In this study, polyvinylidene membranes had the highest ligand binding capacity of 0.22 mg cm(-2) and specific, competitive affinity binding of avidin-peroxidase was shown in the presence of up to 0.2 mg ml(-1) 'contaminant' proteins. The resultant biocompatible affinity chromatographic system was regenerated and reused with no significant change in performance for up to five cycles.
报道了一种用于生物活性抗生物素蛋白标记的酶或多肽生物特异性结合的新型、可重复使用的生物素化亲和色谱策略。使用抗生物素蛋白偶联的过氧化物酶融合蛋白作为测试系统，还展示了非特异性蛋白质屏蔽和基质再生。两亲性表面活性剂普朗尼克F108用作亲和连接体，通过与膜色谱基质非共价结合，而普朗尼克的末端羟基与生物配体生物素共价偶联。合成了具有不同表面化学性质的平面无孔膜，以测试生物素化普朗尼克的基质依赖性亲和结合及其抵抗非特异性蛋白质吸附的各自能力。使用十二烷基硫酸钠（SDS）进行膜再生能够置换吸附的蛋白质和普朗尼克。SDS胶束（34 mM）可有效解吸膜结合蛋白，而5 mM SDS在20℃孵育20小时后可去除高达85%的结合配体。在本研究中，聚偏二氟乙烯膜具有最高的配体结合容量，为0.22 mg cm(-2)，并且在存在高达0.2 mg ml(-1)“污染物”蛋白的情况下，显示出抗生物素蛋白-过氧化物酶的特异性竞争性亲和结合。所得的生物相容性亲和色谱系统可再生和重复使用，在多达五个循环中性能无显著变化。
