人多能干细胞特异性凝集素探针 rBC2LCN 的表达和纯化
Expression and Purification of a Human Pluripotent Stem Cell-Specific Lectin Probe, rBC2LCN.
机构信息
Biotechnology Research Institute for Drug Discovery (BRD), National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan.
出版信息
Methods Mol Biol. 2020;2132:453-461. doi: 10.1007/978-1-0716-0430-4_44.
rBC2LCN is a recombinant N-terminal domain of BC2L-C lectin that is derived from the gram-negative bacteria Burkholderia cenocepacia and specifically binds to Fucα1-2Galβ1-3GlcNAc/GalNAc. Glycome analysis using a high-density lectin microarray revealed that rBC2LCN specifically binds to human pluripotent stem cells (hPSCs) but not to non-hPSCs. The lectin can be added to the cell culture medium for the live staining of hPSCs without causing visible cytotoxicity. Moreover, it can be used in flow cytometric analysis and for the staining of fixed hPSCs. rBC2LCN is a single-chain molecule with a low molecular weight (trimer of 16 kDa), which can be produced in large quantities in Escherichia coli (0.1 g/L). Therefore, rBC2LCN may be a cost-effective probe for use in hPSCs, unlike other hPSC surface marker antibodies that require a mammalian cell expression system for production. In this study, we describe the protocols for the expression and purification of rBC2LCN from E. coli and live staining of hPSCs using this probe.
rBC2LCN 是 BC2L-C 凝集素的重组 N 端结构域，来源于革兰氏阴性细菌洋葱伯克霍尔德菌，特异性结合 Fucα1-2Galβ1-3GlcNAc/GalNAc。使用高密度凝集素微阵列进行糖组学分析表明，rBC2LCN 特异性结合人类多能干细胞（hPSC），但不结合非 hPSC。该凝集素可添加到细胞培养基中，用于 hPSC 的活细胞染色，而不会产生明显的细胞毒性。此外，它可用于流式细胞分析和固定 hPSC 的染色。rBC2LCN 是一种分子量低的单链分子（三聚体 16 kDa），可在大肠杆菌中大量生产（0.1 g/L）。因此，rBC2LCN 可能是一种用于 hPSC 的具有成本效益的探针，与其他需要使用哺乳动物细胞表达系统进行生产的 hPSC 表面标记抗体不同。在这项研究中，我们描述了从大肠杆菌表达和纯化 rBC2LCN 以及使用该探针对 hPSC 进行活细胞染色的方案。
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