title: Combining a PagP fusion protein system with nickel ion-catalyzed cleavage to produce intrinsically disordered proteins in E. coli.
abstract: Many proteins contain intrinsically disordered regions that are highly solvent-exposed and susceptible to post-translational modifications. Studying these protein segments is critical to understanding their physiologic regulation, but proteolytic degradation can make them difficult to express and purify. We have designed a new protein expression vector that fuses the target protein to the N-terminus of the integral membrane protein, PagP. The two proteins are connected by a short linker containing the sequence SRHW, previously shown to be optimal for nickel ion-catalyzed cleavage. The methodology is demonstrated for an intrinsically disordered segment of cardiac troponin I. cTnI[135-209]-SRHW-PagP-His6 fusion protein was overexpressed in Escherichia coli, accumulating in insoluble inclusion bodies. The protein was solubilized, purified using nickel affinity chromatography, and then cleaved with 0.5mM NiSO4 at pH 9.0 and 45 °C, all in 6M guanidine-HCl. Nickel ion-catalyzed peptide bond hydrolysis is an effective chemical cleavage technique under denaturing conditions that preclude the use of proteases. Moreover, nickel-catalyzed cleavage is more specific than the most commonly used agent, cyanogen bromide, which cleaves C-terminal to methionine residues. We were able to produce 15 mg of purified cTnI[135-209] from 1L of M9 minimal media using this protocol. The methodology is more generally applicable to the production of intrinsically disordered protein segments.
authors: Division of General Internal Medicine, Department of Medicine, University of Alberta, Clinical Sciences Building Rm 5-112, Edmonton, AB T6G 2G3, Canada.
authors: Somaya
authors: S
authors: Zahran
authors: Department of Biochemistry, University of Alberta, Medical Sciences Building Rm 4-19, Edmonton, AB T6G 2H7, Canada.
authors: Jonathan S
authors: JS
authors: Pan
authors: Division of General Internal Medicine, Department of Medicine, University of Alberta, Clinical Sciences Building Rm 5-112, Edmonton, AB T6G 2G3, Canada.
authors: Philip B
authors: PB
authors: Liu
authors: Division of General Internal Medicine, Department of Medicine, University of Alberta, Clinical Sciences Building Rm 5-112, Edmonton, AB T6G 2G3, Canada; Department of Biochemistry, University of Alberta, Medical Sciences Building Rm 4-19, Edmonton, AB T6G 2H7, Canada. Electronic address: phwang1@ualberta.ca.
authors: Peter M
authors: PM
authors: Hwang
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D064267:Intrinsically Disordered Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D064267:Intrinsically Disordered Proteins; D011993:Recombinant Fusion Proteins; D009532:Nickel; D000217:Acyltransferases; C468520:PagP protein, E coli
keywords: Chemical cleavage; Escherichia coli; Fusion protein expression; Inclusion bodies; Intrinsically disordered regions; Nickel ion-catalyzed peptide bond hydrolysis