Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin
Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin
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DOI：
10.1074/jbc.m310989200
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发表时间：
2004-03-05
影响因子：
4.8
通讯作者：
Kulomaa, MS
中科院分区：
文献类型：
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作者：
Hytönen, VP;Nyholm, TKM;Kulomaa, MS
蛋白质鸡抗生物素蛋白是在各种应用中常用的工具。抗生物素蛋白基因属于一个基因家族，该家族还包括七个其他成员，称为抗生物素蛋白相关基因（AVR）。我们在这里报告的抗生物素蛋白家族的成员，抗生物素蛋白相关蛋白AVR 4/5的极高的热稳定性和功能特性。使用差示扫描量热仪、微粒分析和微孔板测定来检查AVR 4/5的热稳定性特征。利用等温量热仪和IAsys光学生物传感器研究了其生物素结合特性。根据这些分析，在不存在生物素的情况下，AVR 4/5明显比抗生物素蛋白（T-m = 83.5 +/-0.1 ℃）或细菌链霉抗生物素蛋白（T-m = 75.5 ℃）更稳定（T-m = 107.4 +/-0.3 ℃）。AVR4/5还表现出与抗生物素蛋白和链霉抗生物素蛋白相当的对生物素的高亲和力（K-d约为3.6 × 10（-14）M）（K-d约为10（-15）M）。分子建模和定点突变被用来研究所观察到的高热稳定性背后的分子细节。结果表明，AVR4/5及其突变体具有很高的潜力，作为新的改进的工具的应用程序，特别是高稳定性和紧密的生物素结合的需要。
The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (T-m = 107.4 +/- 0.3 degreesC) than avidin (T-m = 83.5 +/- 0.1 degreesC) or bacterial streptavidin (T-m = 75.5 degreesC). AVR4/5 also exhibits a high affinity for biotin (K-d approximate to 3.6 x 10(-14) M) comparable to that of avidin and streptavidin (K-d approximate to 10(-15) M). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4/5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed.