title: High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.
abstract: The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.
authors.affiliation: Department of Biomedical Engineering, University of Alberta, Edmonton, Alberta, Canada.|National Institute for Nanotechnology, National Research Council, Edmonton, Alberta, Canada.
authors.forename: Markian S
authors.initials: MS
authors.lastname: Bahniuk
authors.affiliation: National Institute for Nanotechnology, National Research Council, Edmonton, Alberta, Canada.|Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Abdullah K
authors.initials: AK
authors.lastname: Alshememry
authors.affiliation: Department of Biomedical Engineering, University of Alberta, Edmonton, Alberta, Canada.|National Institute for Nanotechnology, National Research Council, Edmonton, Alberta, Canada.|Department of Chemical & Materials Engineering, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Larry D
authors.initials: LD
authors.lastname: Unsworth
mesh_terms: D002846:Chromatography, Affinity; D004549:Elastin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D004549:Elastin
keywords: concatemerization; denaturing metal affinity chromatography; elastin-like polypeptides; inverse temperature transitions; marginally soluble protein purification; recursive directional ligation