Development of sulfhydryl-reactive silica for protein immobilization in high-performance affinity chromatography
Development of sulfhydryl-reactive silica for protein immobilization in high-performance affinity chromatography
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DOI：
10.1021/ac061779j
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发表时间：
2007-02-15
影响因子：
7.4
通讯作者：
Hage, David S.
中科院分区：
文献类型：
--
作者：
Mallik, Rangan;Wa, Chunling;Hage, David S.
开发了两种通过巯基将蛋白质和其他配体固定到二氧化硅上的技术。这些方法使用马来酰亚胺活化二氧化硅（SMCC法）或碘乙酰基活化二氧化硅（SIA法）。采用人血清白蛋白（HSA）作为模型蛋白，对所得支架进行了高效亲和层析测试。对正常和碘乙酰酰胺修饰的HSA的研究表明，这些方法对该蛋白上的巯基具有很高的选择性，占该蛋白与马来酰亚胺或碘乙酰基活化二氧化硅偶联率的77-81%。对这些载体的总蛋白含量、结合能力、特异性活性、非特异性结合、稳定性和对几种测试溶质的手性选择性进行了评估。与通过希夫碱法（即胺基偶联技术）固定到二氧化硅的HSA相比，使用马来酰亚胺活化二氧化硅制备的HSA柱具有这些性能的最佳总体结果。在这项工作中开发的支持的一个关键优势是，它们提供了比基于胺的偶联方法更大的位点选择性固定和配体活性的潜力。这些特点使这些支持有吸引力的蛋白质柱的开发等应用，如生物相互作用和手性分离的研究。
Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in high-performance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77-81% of this protein to maleimide- or iodoacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, nonspecific binding, stability, and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations.