title: Purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilin-inspired polypeptides.
abstract: Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature.
authors.affiliation: CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia, QLD 4072, Australia. russell.lyons@csiro.au
authors.forename: Russell E
authors.initials: RE
authors.lastname: Lyons
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Elvin
authors.forename: Karin
authors.initials: K
authors.lastname: Taylor
authors.forename: Nicolas
authors.initials: N
authors.lastname: Lekieffre
authors.forename: John A M
authors.initials: JA
authors.lastname: Ramshaw
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D000852:Anopheles / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D011232:Chemical Precipitation; D003001:Cloning, Molecular / Q000379:methods*; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C030951:resilin; D013917:Thrombin; D000645:Ammonium Sulfate