Specific Labeling of Streptavidin for Better Understanding of Ligand Modification in Modular Method for Affinity Labeling (MoAL)
作者:Munetaka Kunishima、Daiki Kato、Shuichi Nakanishi、Masanori Kitamura、Kohei Yamada、Keiji Terao、Tomoya Asano
DOI:10.1248/cpb.c14-00468
日期:——
We studied the specific labeling of streptavidin using the modular method for affinity labeling (MoAL) that we developed based on a catalytic amide-forming reaction using 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and a tertiary amine catalyst. The primary structures of avidin and streptavidin are significantly different from each other, and streptavidin does not possess an acidic amino acid equivalent to Asp108 of avidin, which is the target acidic amino acid that was labeled using MoAL. However, using biotinylated modular ligand catalysts (MLC) originally designed for labeling avidin, the labeling of streptavidin was found to successfully proceed at Glu51, which is located in a different region. The present study indicates that MoAL is readily applicable to protein labeling without a precise design for MLC. The most important factor for the design of MLC is to ensure that the linker is of sufficient length to connect the ligands to a catalytic site.
我们研究了利用我们开发的模块化亲和标记方法(MoAL)对链霉亲和素进行特定标记的方法,该方法基于使用2-氯-4,6-二甲氧基-1,3,5-三嗪(CDMT)和三级胺催化剂的催化酰胺生成反应。亲和素和链霉亲和素的主要结构差异显著,链霉亲和素没有相当于亲和素中的Asp108的酸性氨基酸,而这个酸性氨基酸正是通过MoAL进行标记的目标。然而,使用最初为标记亲和素设计的生物素化模块化配体催化剂(MLC),我们发现链霉亲和素的标记成功地在Glu51进行,该位置位于不同区域。本研究表明,MoAL可以轻松适用于蛋白质标记,而不需要对MLC进行精确设计。MLC设计中最重要的因素是确保连接链足够长,以将配体连接到催化位点。