Zhang Y, Mabuchi K, Tao T
Muscle Research Group, Boston Biomedical Research Institute, MA 02114, USA.
Biochim Biophys Acta. 1997 Nov 14;1343(1):51-8. doi: 10.1016/s0167-4838(97)00120-9.
The major myosin light chain phosphatase is composed of three subunits with apparent molecular masses of 130, 38 and 20 kDa, corresponding to the myosin-binding, catalytic and a regulatory subunit of unknown function, respectively. In this work, we have amplified the cDNA coding for each of the three subunits by the polymerase chain reaction, and expressed the 130 kDa subunit in insect cells using the baculovirus expression system. Limited chymotrypsin digestion show that the folding of the expressed protein is similar to that in the native holoenzyme. N-Terminal sequencing reveals that our recombinant protein is authentic. Mass spectrometry shows that the expressed protein is full length. The recombinant protein is capable of binding myosin based on the ELISA assay and myosin affinity chromatography. Finally, rotary shadowing electron microscopy reveals an elongated structure with three globular domains connected by flexible strands. These results pave the way for future biochemical, structural and site-directed mutagenesis studies on the myosin light chain phosphatase. We also found that the cDNA of the 20 kDa subunit may code for a smaller protein with a molecular mass of 18.5 kDa.
主要的肌球蛋白轻链磷酸酶由三个亚基组成，其表观分子量分别为130、38和20 kDa，分别对应于肌球蛋白结合亚基、催化亚基和功能未知的调节亚基。在本研究中，我们通过聚合酶链反应扩增了编码这三个亚基的cDNA，并使用杆状病毒表达系统在昆虫细胞中表达了130 kDa的亚基。有限的胰凝乳蛋白酶消化表明，表达蛋白的折叠与天然全酶中的折叠相似。N端测序表明我们的重组蛋白是真实的。质谱分析表明表达的蛋白是全长的。基于酶联免疫吸附测定和肌球蛋白亲和色谱法，重组蛋白能够结合肌球蛋白。最后，旋转阴影电子显微镜揭示了一种细长结构，具有由柔性链连接的三个球状结构域。这些结果为未来对肌球蛋白轻链磷酸酶的生化、结构和定点诱变研究铺平了道路。我们还发现，20 kDa亚基的cDNA可能编码一种分子量为18.5 kDa的较小蛋白。
