Expression and purification of maltose-binding protein fusions.
This unit describes the procedure for subcloning the sequence encoding the protein of interest into an maltose-binding protein (MBP) vector, and expressing and purifying the fusion protein from the cytoplasm. MBP vectors include a sequence that encodes the four-amino-acid recognition site for the specific protease factor Xa. The site is placed so it can be used to separate the protein of interest from MBP after affinity purification. A support protocol provides a pilot experiment for analyzing the solubility, affinity for the amylose resin, and export of a particular fusion protein. An alternate protocol gives instructions for purifying a fusion protein from the periplasm for fusions that are made in the signal sequence vector and are exported. Additional support protocols detail two different chromatographic methods for separating the protein of interest from MBP after factor Xa cleavage.
Riggs
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New England Biolabs, Beverly, Massachusetts, USA.
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005822:Genetic Vectors; D058619:Maltose-Binding Proteins; D019897:Periplasm; D033902:Periplasmic Binding Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins