HomeFAQsMuch of my fusion protein flows through the amylose column. Is there anything I can do to improve my fusion’s affinity for the amylose column?
FAQ: Much of my fusion protein flows through the amylose column. Is there anything I can do to improve my fusion’s affinity for the amylose column?
A MBP fusion protein might not stick to the amylose column because of the presence of some factor in the extract that interferes with binding, or because of a low intrinsic affinity. Factors in the crude extract that can interfere with binding include nonionic detergents and cellular components that are released during alternative methods of lysis (prolonged treatment with lysozyme or multiple passes through a French press). In addition, cells grown in LB and similar media have substantial amounts of an amylase that interferes with binding, presumably by either cutting the fusion off the column or by releasing maltose that elutes the fusion from the column. By including glucose in the media, expression of this amylase is repressed and the problem is alleviated. A low intrinsic affinity could be caused by an interaction between the protein of interest and MBP that either blocks or distorts the maltose-binding site. Although this may be inherent in the protein of interest, sometimes the problem can be alleviated by shortening or lengthening the polypeptide that is fused to MBP.