Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd., Athens, GA 30602, USA.
Department of Chemistry.
Glycobiology. 2021 May 3;31(4):425-435. doi: 10.1093/glycob/cwaa071.
Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins requires enrichment with less abundant, NMR-active, isotopes such as 13C and 15N. This is routine for proteins that can be expressed in bacterial culture where low-cost isotopically enriched metabolic substrates can be used. However, it can be expensive for glycosylated proteins expressed in mammalian culture where more costly isotopically enriched amino acids are usually used. We describe a simple, relatively inexpensive procedure in which standard commercial media is supplemented with 13C-enriched glucose to achieve labeling of all glycans plus all alanines of the N-terminal domain of the highly glycosylated protein, CEACAM1. We demonstrate an ability to detect partially occupied N-glycan sites, sites less susceptible to processing by an endoglycosidase, and some unexpected truncation of the amino acid sequence. The labeling of both the protein (through alanines) and the glycans in a single culture requiring no additional technical expertise past standard mammalian expression requirements is anticipated to have several applications, including structural and functional screening of the many glycosylated proteins important to human health.
为了进行更大的蛋白质的核磁共振（NMR）特征分析，需要对其进行富集，使用较少的丰度、NMR 活性同位素，如 13C 和 15N。这对于可以在细菌培养中表达的蛋白质来说是常规操作，因为可以使用低成本的同位素富集代谢底物。然而，对于在哺乳动物培养中表达的糖基化蛋白质来说，这可能会很昂贵，因为通常使用更昂贵的同位素富集氨基酸。我们描述了一种简单、相对廉价的方法，即在标准商业培养基中添加 13C 富集的葡萄糖，以实现高度糖基化蛋白质 CEACAM1 的所有聚糖和 N 末端结构域中所有丙氨酸的标记。我们证明了能够检测部分占据的 N-糖基化位点、对内糖苷酶处理不太敏感的位点以及一些意想不到的氨基酸序列截断。通过单一培养物对蛋白质（通过丙氨酸）和聚糖进行标记，不需要超出标准哺乳动物表达要求的额外技术专业知识，预计将有多种应用，包括对许多与人类健康相关的糖基化蛋白质的结构和功能进行筛选。
