Improved procedure for direct coupling of carbohydrates to proteins via reductive amination
Improved procedure for direct coupling of carbohydrates to proteins via reductive amination
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DOI：
10.1021/bc800153t
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发表时间：
2008-07-01
影响因子：
4.7
通讯作者：
Allred, Benjamin
中科院分区：
文献类型：
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作者：
Gildersleeve, Jeffrey C.;Oyelaran, Oyindasola;Allred, Benjamin
碳水化合物-蛋白质缀合物广泛用于基础研究，并作为各种细菌疫苗和癌症疫苗的免疫原。因此，已经做出了重大努力来开发用于构建这些缀合物的简单且可靠的方法。虽然通过还原胺化直接偶联是一种有吸引力的方法，但该反应通常非常低效。在本文中，我们报告了改进的反应条件，提供了约500%的产率增加。除了优化一系列标准反应参数外，我们发现添加500 mM硫酸钠可提高偶联效率。为了说明这些条件的效用，产生一系列高甘露糖BSA缀合物并掺入碳水化合物微阵列中。可以观察到配体与ConA的结合，并且使用阵列测量的表观亲和常数（K（d）s）与表面等离子体共振报道的值良好一致。结果表明，该条件适用于微克级反应，与复杂的碳水化合物相容，并产生生物活性缀合物。
Carbohydrate-protein conjugates are utilized extensively in basic research and as immunogens in a variety of bacterial vaccines and cancer vaccines. As a result, there have been significant efforts to develop simple and reliable methods for the construction of these conjugates. While direct coupling via reductive amination is an appealing approach, the reaction is typically very inefficient. In this paper, we report improved reaction conditions providing an approximately 500% increase in yield. In addition to optimizing a series of standard reaction parameters, we found that addition of 500 mM sodium sulfate improves the coupling efficiency. To illustrate the utility of these conditions, a series of high mannose BSA conjugates were produced and incorporated into a carbohydrate microarray. Ligand binding to ConA could be observed and apparent affinity constants (K(d)s) measured using the array were in good agreement with values reported by surface plasmon resonance. The results show that the conditions are suitable for microgram-scale reactions, are compatible with complex carbohydrates, and produce biologically active conjugates.