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 t o the myosin-binding, catalytic and a regulatory subunit of unknown fu nction, respectively. In this work, we have amplified the cDNA coding for each of the three subunits by the polymerase chain reaction, and e xpressed the 130 kDa subunit in insect cells using the baculovirus exp ression system. Limited chymotrypsin digestion show that the folding o f 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. T he recombinant protein is capable of binding myosin based on the ELISA assay and myosin affinity chromatography. Finally, rotary shadowing e lectron 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 o n the myosin light chain phosphatase. We also found that the cDNA of t he 20 kDa subunit may code for a smaller protein with a molecular mass of 18.5 kDa. (C) 1997 Elsevier Science B.V.