L E Petrovskaia, S Sh Gapizov, L N Shingarova, E A Kriukova, E F Boldyreva, S A Iakimov, E V Svirshchevskaia, E P Lukashev, D A Dolgikh, M P Kirpichnikov
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引用次数: 0
摘要
本文描述了一种新型杂交分子,包括红色荧光蛋白 mCherry 和第 10 个 III 型人类纤连蛋白结构域(10Fn3)--一种可用于构建具有不同结合特异性的抗体模拟物的替代支架蛋白。我们构建了编码混合荧光蛋白的不同基因变体,并研究了它们在大肠杆菌细胞中的表达情况。结果表明,mCherry 的 N 端位置和对其 N 端氨基酸序列的修改可促进可溶性混合蛋白的高效细菌表达。在此基础上,我们获得了含有 10Fn3 的 alphaVbeta3 整合素结合变体的杂交荧光蛋白 ChIBF,并证明了用共聚焦显微镜观察 MDCK 上皮细胞表面 alphaVbeta3 整合素的可能性。
[Fluorescent fusion proteins with 10th human fibronectin domain].
In the current paper we describe a new type of hybrid molecules including red fluorescent protein mCherry and 10th type III human fibronectin domain (10Fn3) - one of the alternative scaffold proteins which can be used for the construction of antibody mimics with various binding specificity. We have constructed different gene variants encoding for the hybrid fluorescent protein and studied their expression in Escherichia coli cells. It was shown that N-terminal position of mCherry and modification of its N-terminal amino acid sequence promotes efficientbacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construction we have obtained the hybrid fluorescent protein ChIBF, containing alphaVbeta3-integrin binding vari- ant of 10Fn3, and demonstrated the possibility of its utilization for the visualization of alphaVbeta3-integrin at the surface of MDCK epithelial cells by confocal microscopy.