Molecular cloning of chick cardiac muscle tensin. Full-length cDNA sequence, expression, and characterization.
Molecular cloning of chick cardiac muscle tensin. Full-length cDNA sequence, expression, and characterization.
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发表时间：
1994-09
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通讯作者：
S. Lo;Q. An;S. Bao;W. Wong;Y. Liu;P. Janmey;J. Hartwig;L. Chen
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作者：
S. Lo;Q. An;S. Bao;W. Wong;Y. Liu;P. Janmey;J. Hartwig;L. Chen
本文描述了鸡心肌紧张素7.1千碱基cDNA的分子克隆。它包含1744个氨基酸（aa）残基的开放阅读框。序列分析显示，除了先前提到的SH2结构域（Davis， S., Lu， M. L., Lo， S. H., Lin， S., Butler， J. A., Druker， B. J., Roberts， T. M., An， Q., and Chen， L. B. (1991) Science 252, 712-715）外，张力蛋白包含几乎所有已知的插入蛋白序列（362 aa），这是一种肌动蛋白封盖蛋白，允许肌动蛋白单体被“插入”（Schroer， E., and Wegner， A., 1985）。中国生物医学工程学报（英文版）。此外，张力蛋白与肌动蛋白（30 aa内46.7%的同源性）、β -spectrin的肌动蛋白结合一致性（26 aa内40%的同源性）、BCR （25 aa内40%的同源性）、catenin α （45 aa内35%的同源性）、synapsin Ia （156 aa内25.6%的同源性）、IL-3受体（384 aa内20.2%的同源性）和IL-2/EPO受体（20 aa内14%的同源性）具有部分同源性。用流感衍生表位标记的重组全长张力蛋白，通过杆状病毒系统过表达并纯化到明显的同质性。在sds -聚丙烯酰胺凝胶电泳中，它以200 kda的蛋白迁移，与天然张力蛋白相似。通过光散射、电子显微镜和凝胶过滤表征了张力素分子的结构。制备并鉴定了9种识别张力素不同区域的单克隆抗体。将表位标记的重组紧张素基因亚克隆到pRcCMV载体中，转染NIH 3T3细胞。鸡紧张素特异性单克隆抗体（与小鼠紧张素无交叉反应）的免疫荧光染色显示，表达的蛋白确实像天然紧张素一样定位于局灶接触处。
Here we describe the molecular cloning of 7.1-kilobase cDNA encoding chick cardiac muscle tensin. It contains an open reading frame of 1,744 amino acid (aa) residues. Sequence analysis reveals that, in addition to the previously noted SH2 domain (Davis, S., Lu, M. L., Lo, S. H., Lin, S., Butler, J. A., Druker, B. J., Roberts, T. M., An, Q., and Chen, L. B. (1991) Science 252, 712-715), tensin contains virtually all of the known sequence (362 aa) of insertin, an actin-capping protein that allows actin monomer to be "inserted" (Schroer, E., and Wegner, A. (1985) Eur. J. Biochem. 153, 515-520). Moreover, tensin shares partial homology with actin (46.7% identity in 30 aa), beta-spectrin's actin-binding consensus (40% identity in 26 aa), BCR (40% identity in 25 aa), catenin alpha (35% identity in 45 aa), synapsin Ia (25.6% identity in 156 aa), IL-3 receptor (20.2% identity in 384 aa), and IL-2/EPO receptors (14% identity in 20 aa). Recombinant full-length tensin, tagged with an influenza-derived epitope, was over-expressed by a baculovirus system and purified to apparent homogeneity. It migrates as a 200-kDa protein in SDS-polyacrylamide gel electrophoresis, similar to the native tensin. The structure of the tensin molecule has been characterized by light scattering, electron microscopy, and gel filtration. Nine monoclonal antibodies recognizing different regions of tensin have been prepared and characterized. The epitope-tagged recombinant tensin gene was subcloned into a pRcCMV vector and transfected into NIH 3T3 cells. Immunofluorescence stainings with monoclonal antibodies specific for chick tensin (not cross-reactive with mouse tensin) showed that the expressed protein is indeed localized at focal contacts, as that of native tensin.