增加规则重复序列对光滑鳖甲抗冻蛋白ApAFP914热滞活性的影响

Effect of Regular Repetitive Sequence on the Thermal Hysteresis Activity of Antifreeze Protein ApAFP914 in Anatolica polita

旨在探究光滑鳖甲抗冻蛋白Ap AFP914结构与热滞活性(Thermal hysteresis activity,THA)的关系。采用基因合成的方法,在光滑鳖甲抗冻蛋白基因Ap AFP914中增加单个规则重复序列并进行蛋白原核表达及热滞活性测定。结果显示,增加单个重复序列的Ap AFP-C914为312 bp,融合蛋白Trx A-Ap AFP-C914经SDS-PAGE及Western bolt分析表明,分子量为34 k D。差示扫描量热法(Differential scanning calorimetry,DSC)测定表明,在50μg/m L的浓度下,增加单个重复序列显著提高了Ap AFP的THA活性。光滑鳖甲抗冻蛋白Ap AFP914增加一个重复序列可显著提高其热滞活性。

This work aims to explore the relationship between protein structure and thermal hysteresis activity of antifreeze protein ApAFP914 in Anatolica polita. A regular repetitive sequence was added in the ApAFP914 by gene synthesis. The synthesized gene was then expressed in prokaryotie cells and the thermal hysteresis activity of the protein was determined. Results showed that the gene ApAFP-C914 with a regular repetitive sequence added was 312 bp. The fusion protein TrxA-ApAFP-C914 was verified by SDS-PAGE and Western bolt, and its molecular weight was 34 kD. The measurement by differential scanning calorimetry showed that the thermal hysteresis activity of ApAFP increased significantly （ P〈0.05 ） by adding a regular repetitive sequence at the concentration of 50 -tg/mL. Conclusively, adding repetitive sequence may significantly increase the thermal hysteresis activity of antifreeze protein ApAFP914 in A. polita.