差示扫描量热法分析光滑鳖甲抗冻蛋白ApAFP914 TXT基序对抗冻活性的影响

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

生物技术通报 ›› 2016, Vol. 32 ›› Issue (9): 210-217.doi: 10.13560/j.cnki.biotech.bull.1985.2016.09.028

差示扫描量热法分析光滑鳖甲抗冻蛋白ApAFP914 TXT基序对抗冻活性的影响

杜荣俸, 刘忠渊, 毛新芳

新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,乌鲁木齐 830046

收稿日期:2015-12-10 出版日期:2016-09-25 发布日期:2016-10-10
作者简介:杜荣俸,男,硕士研究生,研究方向：生物化学与分子生物学;E-mail：408832008@qq.com
基金资助:
国家自然科学基金项目（31200588）

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

DU Rong-feng, LIU Zhong-yuan, MAO Xin-fang

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046

Received:2015-12-10 Published:2016-09-25 Online:2016-10-10

摘要/Abstract

摘要： 研究光滑鳖甲抗冻蛋白ApAFP914及其突变体的原核表达及活性,推测TXT基序的突变对昆虫抗冻蛋白抗冻活性的影响。通过定点突变新疆荒漠昆虫光滑鳖甲抗冻蛋白apafp914基因TXT基序的规则位点个数,并亚克隆至pET32a原核表达载体,转化大肠杆菌,Ni-NTA纯化得到融合蛋白TrxA-ApAFP914及3种突变体蛋白;利用SwisS-Model服务器预测分析了ApAFP914蛋白的三维结构;通过差示扫描量热法测定TrxA-ApAFP914及其突变体的热滞活性。结果显示,4种融合蛋白分子量均在30 kD左右;且突变蛋白TrxA-A19T具有最高的热滞活性,而突变体TrxA-T33F和TrxA-T33&45F的热滞活性显著低于未突变的TrxA-914。研究结果表明昆虫抗冻蛋白的TXT基序越规则其具有的热滞活性越高。

关键词: 光滑鳖甲抗冻蛋白, TXT基序, 差示扫描量热法（DSC）

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）

杜荣俸, 刘忠渊, 毛新芳. 差示扫描量热法分析光滑鳖甲抗冻蛋白ApAFP914 TXT基序对抗冻活性的影响[J]. 生物技术通报, 2016, 32(9): 210-217.

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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差示扫描量热法分析光滑鳖甲抗冻蛋白ApAFP914 TXT基序对抗冻活性的影响

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

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