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Combining a PagP fusion protein system with nickel ion-catalyzed cleavage to produce intrinsically disordered proteins in E. coli
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- Author(s) / Creator(s)
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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 E. coli, accumulating in insoluble inclusion bodies. The protein was solubilized, purified using nickel affinity chromatography, and then cleaved with 0.5 mM NiSO4 at pH 9.0 and 50°C, all in 6 M 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 1 L of M9 minimal media using this protocol. The methodology is more generally applicable to the production of intrinsically disordered protein segments.
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- Date created
- 2015-01-01
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- Subjects / Keywords
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- Intrinsically disordered regions
- Inclusion bodies
- Fusion protein expression
- Escherichia coli
- Nickel ion-catalyzed peptide bond hydrolysis
- Chemical cleavage
- PagP
- PhoPQ-activated gene P
- KSI
- Ketosteroid isomerase
- MALDI-TOF
- Matrix-assisted laser desorption ionization time-of-flight
- NMR
- Nuclear magnetic resonance
- IDR
- Intrisically disordered region
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- Type of Item
- Article (Draft / Submitted)