Expression of the phosphorylase kinase gamma subunit catalytic domain in Escherichia coli
Cox, S.; Johnson, L.N.
Protein Engineering 5(8): 811-819
1992
ISSN/ISBN: 0269-2139 PMID: 1287663 Document Number: 397700
the catalytic subunit of phosphorylase b kinase (gamma) and an engineered truncated form (gamma-trc, residues 1-297) have been expressed in Escherichia coli. The truncated protein included the entire catalytic domain as defined by sequence alignment with other protein kinases but lacked the putative calmodulin binding domain. Full-length protein was produced in insoluble aggregates. Some activity was regenerated by solubilization in urea and dilution into renaturating buffer but the activity was found to be associated with a smaller molecular weight component. Full-length protein could not be refolded successfully. The truncated gamma subunit was produced in the soluble fraction of the cell as well as in inclusion bodies. The insoluble protein was refolded by dilution from urea and purified to homogeneity, in a one step separation on DEAE-Sepharose to give a protein mol. wt 32 000 +- 2000 with a high sp. act. of 5.3 mu-mol 32P incorporated into phosphorylase b(PPB)/min/nmol. Kinetic parameters gave K-m for ATP 46 +- 3 mu-M and K-m for PPb 27 +- 1 mu-M. The sp. act. and the K-m values are comparable to those observed for the activated holoenzyme and indicate that the gamma-trc retains the substrate recognition and catalytic properties. The ratio of activities at pH 6.8/8.2 was 0.84. gamma-trc was inhibited by ADP with a K-i of 52 mu-M and was sensitive to activation by Mg-2+ and inhibition by Mn-2+, properties that are characteristic of the holoenzyme and the isolated gamma subunit. Calmodulin which confers calcium sensitivity on the isolated gamma subunit had no effect on the enzymic properties of gamma-trc. A small inhibition by free Ca-2+ was observed with peptide substrate.