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TACO cDNA的克隆、表达及TACO的F肌动蛋白交联作用 被引量：1

Cloning and Expression of TACO cDNA Gene in E.coli and TACO's F-actin Cross-linking Ability
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摘要 为进一步研究TACO(tryptophan-aspartate-containing coat protein)的性质、功能及它与肌动蛋白之间的相互关系,利用逆转录PCR技术扩增出TACO的cDNA序列,并将其克隆到pGEX2T表达质粒上.重组表达质粒pGEX2TTACO在宿主菌BL21中得到稳定表达.表达产物经GlutathioneSepharoseTM亲和纯化得到融合蛋白GSTTACO.肌动蛋白纤维(Factin)结合共沉淀实验证明,融合蛋白GSTTACO保持了天然TACO蛋白的F肌动蛋白结合活性.利用肌动蛋白纤维交联(Factincrosslinking)实验和电镜技术发现,纯化后的GSTTACO蛋白具有交联肌动蛋白纤维并将其捆聚成束的能力.实验结果表明TACO很可能起组织者的作用,通过与肌动蛋白纤维的交联,将肌动蛋白纤维捆聚成束从而形成新的肌动蛋白细胞骨架. To further investigate the properties and biological functions of TACO and the interaction between this protein and actin, the TACO eDNA obtained by RT-PCR was inserted into pGEX-2T vector and successfully expressed in E. coli BL21. The fusion protein GST-TACO was purified to a single band（57 kD） on SDS-PAGE from lysate by glutathione-Sepharose^TM. F-actin co-sedimentation assay showed that the purified GST-TACO owned the F-actin binding ability of native TACO. By F-actin cross-linking analysis and electron microscopy imaging, GST-TACO was found to cross-link F-actin into bundles. These results indicate that TACO may act as an organizer to cross-link F-actin into new actin cytoskeletons.
作者 刘长振 李湘辉 隋森芳
机构地区 清华大学生物科学与技术系
出处 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2005年第6期770-774,共5页 Chinese Journal of Biochemistry and Molecular Biology
基金 国家"973"项目(No.2004CB720005)~~
关键词 TACO 克隆与表达 肌动蛋白纤维交联 交联作用 TACO, clone and expression, F-actin cross-linking
分类号 Q78 [生物学—分子生物学] Q245 [生物学—细胞生物学]
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参考文献11

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同被引文献19

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中国生物化学与分子生物学报
2005年 第6期

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