Expression in insect cells and characterization of the 110 kDa anchoring subunit of myosin light chain phosphatase.
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.
Zhang
Y
Y
Muscle Research Group, Boston Biomedical Research Institute, MA 02114, USA.
Mabuchi
K
K
Tao
T
T
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D007313:Insecta; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase