title: A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag.
abstract: We describe a generic protocol for the overproduction and purification of recombinant proteins in Escherichia coli. The strategy utilizes a dual His6-maltose binding protein (HisMBP) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. The MBP moiety serves to enhance the solubility and promote the proper folding of its fusion partners, and the polyhistidine tag facilitates its purification to homogeneity. This protocol is divided into three stages, each of which takes approximately 1 week to complete: (i) construction of a HisMBP fusion vector; (ii) a pilot experiment to assess the yield and solubility of the target protein; and (iii) the large-scale production and purification of the target protein.
authors.affiliation: Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, P.O. Box B, Frederick, Maryland 21702-1201, USA.
authors.forename: Sreedevi
authors.initials: S
authors.lastname: Nallamsetty
authors.forename: David S
authors.initials: DS
authors.lastname: Waugh
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D058619:Maltose-Binding Proteins; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins