title: Cloning and expression of a novel antifreeze protein AFP72 from the beetle Tenebrio molitor.
abstract: A novel antifreeze protein AFP72 cDNA (GenBbank accession No. AY929389) was obtained by RT-PCR from Tenebrio molitor. The 216 bp fragment encodes a protein of 72 amino acid residues. Sequence analysis revealed that the cDNA displays a high degree of homology with T. molitor antifreeze proteins, ranging up to 90.78%. Recombinant plasmids pMAL-p2X-afp72 and pMAL-c2X-afp72 were transferred into E. coil TBI to induce a MBP fusion protein by IPTG. The target fusion protein was released from the periplasm and cytoplasm by the cold osmotic shock procedure and sonication respectively. The content of the fusion protein came up to 38.9 and 41.5% of the total dissolved protein, respectively. The fusion protein was purified through an amylose affinity column, and incised by factor Xa. Molecular sieve chromatography was used to achieve a high state of purity of the target protein. The purified target protein displayed a single band in SDS-PAGE. The fusion protein was shown to increase resistance to low temperatures in bacteria. This finding could help in further investigations of the properties and function of antifreeze proteins.
authors.affiliation: Department of Life Science and Technology, Xinxiang Medical University, Xinxiang Henan 453003, P.R. China. yqh3499@yahoo.com.cn
authors.forename: Qing-Hua
authors.initials: QH
authors.lastname: Yan
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Qing
authors.initials: Q
authors.lastname: Wang
authors.forename: Hui-Rong
authors.initials: HR
authors.lastname: Zhang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013712:Tenebrio / Q000235:genetics / Q000378:metabolism*
chemical_list: D021301:Antifreeze Proteins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins