Maina C V, Riggs P D, Grandea A G, Slatko B E, Moran L S, Tagliamonte J A, McReynolds L A, Guan C D
New England Biolabs, Beverly, MA 01915.
Gene. 1988 Dec 30;74(2):365-73. doi: 10.1016/0378-1119(88)90170-9.
A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.
已构建了一种质粒载体，该载体可指导在大肠杆菌中合成高水平（约占细胞总蛋白的2%）的靶蛋白与麦芽糖结合蛋白（MBP）的融合蛋白。MBP结构域用于通过亲和色谱法将融合蛋白一步纯化至交联的直链淀粉树脂上。融合蛋白在两个结构域之间包含凝血因子Xa蛋白酶的识别序列（异亮氨酸-谷氨酸-甘氨酸-精氨酸）。用因子Xa切割可分离两个结构域，然后通过重复亲和色谱步骤，可将靶蛋白结构域与MBP结构域分离并纯化。通过该方法已成功纯化了一种原核蛋白（β-半乳糖苷酶）和一种真核蛋白（副肌球蛋白）。
