title: Thermolabile antifreeze protein produced in Escherichia coli for structural analysis.
abstract: The only hyperactive antifreeze protein (AFP) found to date in fishes is an extreme variant of the 3-kDa, alpha-helical, alanine-rich type I AFP, which is referred to here as type Ih. Purification of the 33-kDa homodimeric AFP Ih from a natural source was hampered by its low levels in fish plasma; by the need to remove the more abundant smaller isoforms; and by its extreme thermolability. Moreover, ice affinity as a purification tool was spoiled by the tendency of fish IgM antibodies to bind to ice in the presence of AFPs. In order to produce enough protein for crystallography we expressed AFP Ih as a recombinant protein in the Arctic Express® strain of Escherichia coli at 12 °C, just below the thermal denaturation temperature of 16-18 °C. His-tags were not useful because they compromised the activity and yield of AFP Ih. But in the absence of fish antibodies we were able to recover 10-mg quantities of the antifreeze protein using two cycles of ice affinity purification followed by anion-exchange chromatography to remove contaminating chaperones. The purified recombinant AFP Ih yielded diffraction-quality crystals with an extremely asymmetrical unit cell. By transferring the genes of the chaperones into a methionine auxotroph we were able to grow this host at low temperatures and produce sufficient selenomethionine-labeled AFP Ih for crystallography.
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6.
authors.forename: Feng-Hsu
authors.initials: FH
authors.lastname: Lin
authors.forename: Tianjun
authors.initials: T
authors.lastname: Sun
authors.forename: Garth L
authors.initials: GL
authors.lastname: Fletcher
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000818:Animals; D021322:Antifreeze Proteins, Type I / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics*; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005432:Flounder / Q000378:metabolism*; D007053:Ice; D011489:Protein Denaturation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D021322:Antifreeze Proteins, Type I; D029941:Fish Proteins; D007053:Ice; D011994:Recombinant Proteins