title: Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma               antarctica PI12.
abstract: The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia. azafazlin@gmail.com
authors.forename: Noor Haza Fazlin
authors.initials: NH
authors.lastname: Hashim
authors.forename: Izwan
authors.initials: I
authors.lastname: Bharudin
authors.forename: Douglas Law Sie
authors.initials: DL
authors.lastname: Nguong
authors.forename: Sakura
authors.initials: S
authors.lastname: Higa
authors.forename: Farah Diba Abu
authors.initials: FD
authors.lastname: Bakar
authors.forename: Sheila
authors.initials: S
authors.lastname: Nathan
authors.forename: Amir
authors.initials: A
authors.lastname: Rabu
authors.forename: Hidehisa
authors.initials: H
authors.lastname: Kawahara
authors.forename: Rosli Md
authors.initials: RM
authors.lastname: Illias
authors.forename: Nazalan
authors.initials: N
authors.lastname: Najimudin
authors.forename: Nor Muhammad
authors.initials: NM
authors.lastname: Mahadi
authors.forename: Abdul Munir Abdul
authors.initials: AM
authors.lastname: Murad
mesh_terms: D021301:Antifreeze Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001487:Basidiomycota / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003080:Cold Temperature; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D015003:Yeasts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D021301:Antifreeze Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins