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 Ap AFP914 from Anatolica polita borealis Using Differential Scanning Calorimetry

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。

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 Anatoli#a polita borealis, then the mutant gene was cloned into pET32a vector, and expressed in Escherichia coli BL21 （ DE3 ）. The fusionprotein 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.