Position: Home > Articles > Analysis of the TXT Motifs' Effect on the Antifreeze Activity of Antifreeze Protein Ap AFP914 from Anatolica polita borealis Using Differential Scanning Calorimetry
Biotechnology Bulletin
2016,32
(9)
210-217
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
作 者：
杜荣俸;刘忠渊;毛新芳
单 位：
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
关键词：
光滑鳖甲抗冻蛋白;TXT基序;差示扫描量热法(DSC)
摘 要：
研究光滑鳖甲抗冻蛋白Ap AFP914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p ET32a原核表达载体,转化大肠杆菌,Ni-NTA纯化得到融合蛋白Trx A-Ap AFP914及3种突变体蛋白;利用Swis S-Model服务器预测分析了Ap AFP914蛋白的三维结构;通过差示扫描量热法测定Trx A-Ap AFP914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 k D左右;且突变蛋白Trx A-A19T具有最高的热滞活性,而突变体Trx A-T33F和Trx A-T33&45F的热滞活性显著低于未突变的Trx A-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。
译 名：
Analysis of the TXT Motifs' Effect on the Antifreeze Activity of Antifreeze Protein Ap AFP914 from Anatolica polita borealis Using Differential Scanning Calorimetry
作 者：
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;
关键词：
antifreeze protein in Anatolica polita borealis;;TXT motifs;;differential scanning calorimetry(DSC)
摘 要：
This work is to study the prokaryotic expression and activities of antifreeze protein Ap AFP914 and its mutants,anddeduce the effects of mutations of TXT motifs on the insect's antifreeze protein's activities. By site directed mutagenesis in regular sitenumber of TXT motifs of gene apafp914 in Anatolica polita borealis,then the mutant gene was cloned into p ET32 a vector,and expressed inEscherichia coli BL21(DE3). The fusion protein Trx A-Ap AFP914 and the other three mutant proteins were purified using Ni-NTA. The threedimensional structure of the protein Ap AFP914 was predicted and analyzed using Swis S-Model server. The thermal hysteresis activities of Trx AAp AFP914's and its mutants were detected by differential scanning calorimetry(DSC). The results showed that the molecular weight of the 4fusion proteins was about 30 k D,and the mutant protein Trx A-A19 T had the highest thermal hysteresis activity,while the thermal hysteresisactivities of mutant Trx A-T33&45F and Trx A-T33 F were significantly lower than un-mutated Trx A-914. The results indicate that the moreregular the TXT motif of insect antifreeze protein is,the stronger the thermal hysteresis activity of it is.
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