title: Rescuing aggregation-prone proteins in Escherichia coli with a dual His₆-MBP tag.
abstract: Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely recognized as a premier solubilizing agent. In this chapter, we describe how to construct dual His6-MBP-tagged fusion proteins by Gateway(®) recombinational cloning and how to predict their yield and solubility. We also describe a simple and rapid procedure to test the ability of a His6-MBP fusion protein to bind to Ni-NTA resin and to be digested by tobacco etch virus (TEV) protease, along with a method to assess the solubility of the target protein after it has been separated from His6-MBP.
authors.affiliation: Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, B, Frederick, MD, 21702-1201, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Needle
authors.forename: David S
authors.initials: DS
authors.lastname: Waugh
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D058619:Maltose-Binding Proteins / Q000737:chemistry*; D008967:Molecular Biology / Q000379:methods*; D009842:Oligopeptides / Q000737:chemistry; D066329:Protein Aggregates / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
chemical_list: C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease