title: Preparation of Soluble Proteins from Escherichia coli.
abstract: Purification of human IL-1β is used in this unit as an example of the preparation of a soluble protein from E. coli. Bacteria containing IL-1β are lysed, and IL-1 β in the resulting supernatant is purified by anion-exchange chromatography, salt precipitation, and cation-exchange chromatography, and then concentrated. Finally, the IL-1 β protein is applied to a gel-filtration column to separate it from remaining higher- and lower-molecular-weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state. In addition, the purification procedure serves as an example of how to use classical protein purifications methods, which may also be used in conjunction with the affinity-based methods now more commonly used.
authors.affiliation: Protein Expression Laboratory, NIAMD/NIH, Bethesda, Maryland.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Wingfield
mesh_terms: D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053583:Interleukin-1beta / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: C508585:IL1B protein, human; D053583:Interleukin-1beta; D011994:Recombinant Proteins
keywords: interleukin-1beta; protein purification; recombinant protein