title: Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae).
abstract: Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53°C±0.53°C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34°C±0.25°C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.
authors: Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany. christiane.uhlig@awi.de
authors: Christiane
authors: C
authors: Uhlig
authors: Johannes
authors: J
authors: Kabisch
authors: Gottfried J
authors: GJ
authors: Palm
authors: Klaus
authors: K
authors: Valentin
authors: Thomas
authors: T
authors: Schweder
authors: Andreas
authors: A
authors: Krell
mesh_terms: D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003081:Cold Climate; D003080:Cold Temperature; D003460:Crystallization; D017377:Diatoms / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D005615:Freezing; D046448:Ice Cover; D002479:Inclusion Bodies / Q000737:chemistry; D010957:Plasmids; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054712:Salinity; D014169:Transformation, Bacterial
chemical_list: D021301:Antifreeze Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins