Protein Expression and Modification, New England Biolabs, Ipswich, USA.
Department of Microbiology, Stress Adaptation and Metabolism in Enterobacteria Unit, UMR CNRS 2001, Institut Pasteur, Paris, France.
Protein Sci. 2021 Jun;30(6):1235-1246. doi: 10.1002/pro.4088.
Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross-reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co-crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S-transferase protein were engineered in order to identify the smallest fragment still recognized by the α-MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino-acid tag.
麦芽糖结合蛋白（MBP）在重组蛋白表达中用作亲和性和可溶性标签。单克隆抗体 B48 与 MBP 紧密结合，并且在大肠杆菌裂解物中与其他蛋白质没有交叉反应。这种高度特异性表明 MBP 含有一个表位，可作为在大肠杆菌中表达的蛋白质的纯化和可视化标签证明有用。为了发现 MBP 表位，确定了与抗体结合的 MBP 的共晶结构，并且鉴定了 MBP 的四个氨基酸对结合相互作用至关重要。为了鉴定仍然被α-MBP 抗体识别的最小片段，设计了 MBP 的各种片段与谷胱甘肽 S-转移酶蛋白的融合。通过突变工程稳定表位导致最小化的 14 个氨基酸标签。
