In vitro refolding of heterodimeric CapZ expressed in E. coli as inclusion body protein.
CapZ is a heterodimeric Ca(2+)-independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striated muscle cells. It caps the barbed end of actin filaments and promotes nucleation of actin polymerization, thereby regulating actin filament length. Here we report the expression of the two muscle-specific isoforms alpha2 and beta1, 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 were obtained both by simultaneous refolding of urea-solubilized subunits and by rapid dilution into a buffer containing 20% glycerol, 5 mM EGTA, 2 mM DTT, 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 ultrafiltration. Biochemical characterization of the recombinant heterodimer revealed actin binding activities indistinguishable from those of native CapZ as purified from chicken skeletal muscle. Using the same protocol, we were able to refold the beta1, but not the alpha2 isoform as a single polypeptide, indicating a role for beta1 as a molecular template for the folding of alpha2. The reported recombinant approach leads to high yields of active heterodimer and allows the renaturation and characterization of the beta subunit.
Remmert
K
K
Biochemical Cell Biology Group, University of Bielefeld, Bielefeld, 33615, Germany.
Vullhorst
D
D
Hinssen
H
H
D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D051347:CapZ Actin Capping Protein; D002645:Chickens; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000737:chemistry; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020673:Protein Renaturation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D000199:Actins; D051347:CapZ Actin Capping Protein; D017931:DNA Primers; D008840:Microfilament Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins