In vitro refolding of heterodimeric CapZ expressed in E-coli as inclusion body protein

CapZ is a heterodimeric Ca2+-independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striate d muscle cells. It caps the barbed end of actin filaments and promotes nucl eation of actin polymerization, thereby regulating actin filament length. H ere we report the expression of the two muscle-specific isoforms alpha 2 an d beta 1, from chicken in Escherichia coli as individual subunits using the pQE60 expression vector and the subsequent renaturation of the functional CapZ heterodimer from inclusion bodies, Optimal renaturation conditions wer e obtained both by simultaneous refolding of urea-solubilized subunits and by rapid dilution into a buffer containing 20% glycerol, 5 mM EGTA, 2 mM DT T, 1 mM PMSF, and 100 mM. Tris, pH 7.4, The refolding mixture was incubated for 24 h at 15 degrees C and the protein was concentrated by ultrafiltrati on, Biochemical characterization of the recombinant heterodimer revealed ac tin binding activities indistinguishable from those of native CapZ as purif ied from chicken skeletal muscle. Using the same protocol, we were able to refold the beta 1, but not the alpha 2 isoform as a single polypeptide, ind icating a role for beta 1 as a molecular template for the folding of alpha 2. The reported recombinant approach leads to high yields of active heterod imer and allows the renaturation and characterization of the beta subunit. (C) 2000 Academic Press.