title: High-throughput purification of hexahistidine-tagged proteins expressed in E. coli.
abstract: This chapter describes a method for efficient high-throughput purification of hexahistidine-tagged proteins that are expressed in Escherichia coli (E. coli) using immobilized metal affinity chromatography (IMAC) in a 96-well format. This approach is particularly suitable for proteomic applications that require modest amounts of highly purified proteins to be generated very efficiently. This approach is also very useful for identifying protein targets that are most amenable to scaled-up production for use in structural studies. The typical yield of proteins purified using this system is 50-150 microg, which is generally greater than that of many in vitro expression systems and much less costly. The method as described has been optimized for purifying approx 150 microg of hexahistidine-tagged protein, but the method is flexible, so that the amount of affinity matrix and culture volumes can be adjusted for optimal binding capacity and consequently highest purity. Although the method detailed here uses IMAC to purify hexahistidine-tagged proteins, this basic platform can be used with many other tags and affinity resins.
authors.affiliation: Proteomics Group, DOE Joint Genome Institute, Walnut Creek, CA, USA.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Murphy
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Doyle
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D009842:Oligopeptides; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; D006639:Histidine