Analysis of the TXT Motifs’Effect on the Antifreeze Activity of Antifreeze Protein ApAFP914 from Anatolica polita borealis Using Differential Scanning Calorimetry

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.028

Abstract

Abstract: This work is to study the prokaryotic expression and activities of antifreeze protein ApAFP914 and its mutants,and deduce the effects of mutations of TXT motifs on the insect’s antifreeze protein’s activities. By site directed mutagenesis in regular site number of TXT motifs of gene apafp914 in Anatolica polita borealis,then the mutant gene was cloned into pET32a vector,and expressed in Escherichia coli BL21（DE3）. The fusion protein TrxA-ApAFP914 and the other three mutant proteins were purified using Ni-NTA. The three-dimensional structure of the protein ApAFP914 was predicted and analyzed using SwisS-Model server. The thermal hysteresis activities of TrxA-ApAFP914’s and its mutants were detected by differential scanning calorimetry（DSC）. The results showed that the molecular weight of the 4 fusion proteins was about 30 kD,and the mutant protein TrxA-A19T had the highest thermal hysteresis activity,while the thermal hysteresis activities of mutant TrxA-T33&45F and TrxA-T33F were significantly lower than un-mutated TrxA-914. The results indicate that the more regular the TXT motif of insect antifreeze protein is,the stronger the thermal hysteresis activity of it is.

Key words: antifreeze protein in Anatolica polita borealis, TXT motifs, differential scanning calorimetry（DSC）

DU Rong-feng, LIU Zhong-yuan, MAO Xin-fang. Analysis of the TXT Motifs’Effect on the Antifreeze Activity of Antifreeze Protein ApAFP914 from Anatolica polita borealis Using Differential Scanning Calorimetry[J]. Biotechnology Bulletin, 2016, 32(9): 210-217.

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