title: Characterization of a novel β-helix antifreeze protein from the desert beetle Anatolica polita.
abstract: Many ectotherms organisms produce antifreeze proteins (AFPs) which inhibit the growth of ice by binding to the surface of ice crystals. In this study, a novel antifreeze protein gene from the desert beetle Anatolica polita (named as Apafp752) was expressed in a high level in Escherichia coli strain BL21 (DE3). An approximately 30kDa fusion protein thioredoxin (Trx)-ApAFP752 was purified through Ni-NTA affinity chromatography and gel filtration chromatography. The activity of the purified fusion protein Trx-ApAFP752 was analyzed by thermal hysteresis activity (THA) and cryoprotection assay. The results suggested that Trx-ApAFP752 conferred freeze resistance on bacterium in a concentration- and time-dependent manner and the cryoprotective effect increased under alkaline conditions. Circular Dichroism (CD) spectrum analysis showed that the recombinant protein of ApAFP752 possessing β-sheet as the main structure was stable under a wide range of pH from 2.0 to 11.0 and thermal stability below 50°C. The predicted 3D structure showed that Trx-ApAFP752 could form a β-helix structure on the antifreeze protein part, which placed most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi, China. lzy1168@gmail.com
authors.forename: Xinfang
authors.initials: X
authors.lastname: Mao
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Liu
authors.forename: Ji
authors.initials: J
authors.lastname: Ma
authors.forename: Hai
authors.initials: H
authors.lastname: Pang
authors.forename: Fuchun
authors.initials: F
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002942:Circular Dichroism / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D001517:Coleoptera; D003451:Cryoprotective Agents / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007053:Ice; D019476:Insect Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry
chemical_list: D021301:Antifreeze Proteins; D003451:Cryoprotective Agents; D007053:Ice; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins