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

Remmert K, Vullhorst D, Hinssen H (2000)
PROTEIN EXPRESSION AND PURIFICATION 18(1): 11-19.

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CapZ is a heterodimeric Ca2+-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 alpha 2 and 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 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 beta 1, but not the alpha 2 isoform as a single polypeptide, indicating 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 heterodimer and allows the renaturation and characterization of the beta subunit. (C) 2000 Academic Press.
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Remmert K, Vullhorst D, Hinssen H. In vitro refolding of heterodimeric CapZ expressed in E-coli as inclusion body protein. PROTEIN EXPRESSION AND PURIFICATION. 2000;18(1):11-19.
Remmert, K., Vullhorst, D., & Hinssen, H. (2000). In vitro refolding of heterodimeric CapZ expressed in E-coli as inclusion body protein. PROTEIN EXPRESSION AND PURIFICATION, 18(1), 11-19.
Remmert, K., Vullhorst, D., and Hinssen, H. (2000). In vitro refolding of heterodimeric CapZ expressed in E-coli as inclusion body protein. PROTEIN EXPRESSION AND PURIFICATION 18, 11-19.
Remmert, K., Vullhorst, D., & Hinssen, H., 2000. In vitro refolding of heterodimeric CapZ expressed in E-coli as inclusion body protein. PROTEIN EXPRESSION AND PURIFICATION, 18(1), p 11-19.
K. Remmert, D. Vullhorst, and H. Hinssen, “In vitro refolding of heterodimeric CapZ expressed in E-coli as inclusion body protein”, PROTEIN EXPRESSION AND PURIFICATION, vol. 18, 2000, pp. 11-19.
Remmert, K., Vullhorst, D., Hinssen, H.: In vitro refolding of heterodimeric CapZ expressed in E-coli as inclusion body protein. PROTEIN EXPRESSION AND PURIFICATION. 18, 11-19 (2000).
Remmert, K, Vullhorst, D, and Hinssen, Horst. “In vitro refolding of heterodimeric CapZ expressed in E-coli as inclusion body protein”. PROTEIN EXPRESSION AND PURIFICATION 18.1 (2000): 11-19.
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