title: Expression and purification of a nanostructure-forming peptide.
abstract: Peptides have recently attracted interest as building blocks for the assembly of novel functional materials including switchable surfactants, nanocoatings, hydrogels and aqueous vesicles. We expressed a beta-sheet forming peptide that has been widely studied in self-assembly processing, P(11)-2, as a monomer, dimer, tetramer and nonamer fused to an insoluble expression partner, ketosteroid isomerase, using minimal media. Expression was followed by whole cell extraction and isolation of the fusion protein to greater than 90% purity via a single immobilised metal affinity chromatography (IMAC) step. Peptides were chemically cleaved from each other and from the fusion partner, followed by acetone precipitation of the contaminating protein fragments. Pure peptide was recovered by reversed-phase HPLC. The expression level of the fusion protein decreased as the peptide concatamer number increased, as did the efficiency of the chemical cleavage, making the single-peptide process the most efficient overall. Applying this laboratory process to the single-peptide fusion protein nevertheless resulted in a pure peptide yield of greater than 30% of the expressed peptide.
authors.affiliation: Centre for Biomolecular Engineering, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: B M
authors.initials: BM
authors.lastname: Hartmann
authors.forename: W
authors.initials: W
authors.lastname: Kaar
authors.forename: R J
authors.initials: RJ
authors.lastname: Falconer
authors.forename: B
authors.initials: B
authors.lastname: Zeng
authors.forename: A P J
authors.initials: AP
authors.lastname: Middelberg
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000502:physiology*; D049329:Nanostructures / Q000737:chemistry*; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins