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首页> 外文学位 >Construction of recombinant rabbit uterine smooth muscle myosin light chain kinase cDNA for expression in insect cells.
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Construction of recombinant rabbit uterine smooth muscle myosin light chain kinase cDNA for expression in insect cells.

机译：重组兔子宫平滑肌肌球蛋白轻链激酶cDNA在昆虫细胞中表达的构建。

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摘要

Myosin light chain kinases (MLCKs) are a group of enzymes that catalyze the transfer of gamma-phosphate from MgATP to a serine residue near the N-terminus of myosin regulatory light chain (RLC) (Kamm and Stull 2001). Smooth muscle MLCK (smMLCK) is expressed in all adult tissues including skeletal and non-muscle tissues (Kamm and Stull, 2001) where smMLCK phosphorylates only smooth muscle RLC as its substrate (Leachman et al., 1992). For a better understanding of the mechanism of action of smMLCK, chimeric MLCKs were previously constructed in our lab by Pondugula (2004) and stable cell lines expressing wild type and chimeric proteins were established by Kandlur (2006). Recently, sequencing of the smMLCK clone in pCMV-5 we obtained from Dr. J. T. Stull (UT Southwestern Medical Center, Dallas, TX) which was used for all chimera constructs, revealed unwanted mutations and deletions in the 5' terminus. Therefore, construction of the wild type rabbit uterine smooth muscle MLCK expression vector has become essential to reconstruct the chimeras by domain swapping. The goal of this research was to construct recombinant rabbit uterine smooth muscle myosin light chain kinase (smMLCK) cDNA from total RNA isolated from frozen rabbit uterine muscle for expression in an insect cell host system. The construct was verified for expression of rabbit uterine myosin light chain kinase protein by performing transient transfection into the High Five(TM) insect cell expression system (Invitrogen(TM)) and western blot analysis using a monoclonal anti MLCK anti body. Expressed protein has a C-terminus his-tag facilitating easy one step purification by nickel affinity chromatography.
机译：肌球蛋白轻链激酶（MLCKs）是一组酶，可催化从MgATP向肌球蛋白调节性轻链（RLC）N末端附近的丝氨酸残基转移γ-磷酸（Kamm and Stull 2001）。平滑肌MLCK（smMLCK）在包括骨骼和非肌肉组织在内的所有成年组织中表达（Kamm和Stull，2001），其中smMLCK仅磷酸化平滑肌RLC作为底物（Leachman等，1992）。为了更好地了解smMLCK的作用机理，Pondugula（2004）先前在我们的实验室中构建了嵌合MLCKs，Kandlur（2006）建立了表达野生型和嵌合蛋白的稳定细胞系。最近，我们从J.T. Stull博士（德克萨斯西南医学中心，达拉斯，得克萨斯州）获得了pCMV-5中smMLCK克隆的序列，该序列用于所有嵌合体构建体，揭示了5'末端的不需要的突变和缺失。因此，构建野生型兔子宫平滑肌MLCK表达载体对于通过结构域交换重建嵌合体已经变得至关重要。这项研究的目标是从分离自冷冻兔子宫肌的总RNA中构建重组兔子宫平滑肌肌球蛋白轻链激酶（smMLCK）cDNA，以在昆虫细胞宿主系统中表达。通过执行瞬时转染到High Five™昆虫细胞表达系统（Invitrogen™）和使用单克隆抗MLCK抗体进行的蛋白质印迹分析，验证了该构建体可表达兔子宫肌球蛋白轻链激酶蛋白。表达的蛋白质具有一个C末端的his-tag，可通过镍亲和色谱法方便地一步纯化。

著录项

  • 作者

    Noonepalle, Satishkumar.;

  • 作者单位

    Stephen F. Austin State University.;

  • 授予单位 Stephen F. Austin State University.;
  • 学科 Biology Molecular.
  • 学位 M.S.
  • 年度 2008
  • 页码 109 p.
  • 总页数 109
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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