Hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in Escherichia coli.
Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinatorially-tagged His(6)MBP fusion proteins by recombinational cloning and how to evaluate their yield and solubility. We also describe a procedure to determine how efficiently a His(6)MBP fusion protein is cleaved by tobacco etch virus (TEV) protease in E. coli and a method to assess the solubility of the target protein after it has been separated from His(6)MBP.
Austin
Brian P
BP
Macromolecular Crystallography Laboratory, Center for CancerResearch, National Cancer Institute at Frederick, Frederick, MD, USA.
Nallamsetty
Sreedevi
S
Waugh
David S
DS
D000818:Animals; D019677:Artificial Gene Fusion; D002352:Carrier Proteins / Q000235:genetics*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry*; D058619:Maltose-Binding Proteins; D009842:Oligopeptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013010:Sonication
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease