title: A PagP fusion protein system for the expression of intrinsically disordered proteins in Escherichia coli.
abstract: PagP, a beta-barrel membrane protein found in Gram-negative bacteria, expresses robustly in inclusion bodies when its signal sequence is removed. We have developed a new fusion protein expression system based on PagP and demonstrated its utility in the expression of the unstructured N-terminal region of human cardiac troponin I (residues 1-71). A yield of 100mg fusion protein per liter M9 minimal media was obtained. The troponin I fragment was removed from PagP using cyanogen bromide cleavage at methionine residues followed by nickel affinity chromatography. We further demonstrate that optimal cleavage requires complete reduction of methionine residues prior to cyanogen bromide treatment, and this is effectively accomplished using potassium iodide under acidic conditions. The PagP-based fusion protein system is more effective at targeting proteins into inclusion bodies than a commercially available system that uses ketosteroid isomerase; it thus represents an important advance for producing large quantities of unfolded peptides or proteins in Escherichia coli.
authors: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada. pmhwang@gmail.com
authors: Peter M
authors: PM
authors: Hwang
authors: Jonathan S
authors: JS
authors: Pan
authors: Brian D
authors: BD
authors: Sykes
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003488:Cyanogen Bromide / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D019210:Troponin I; D000217:Acyltransferases; C468520:PagP protein, E coli; D003488:Cyanogen Bromide