Figure 3.

Purification of recombinant LeCPK1 and characterization of the autokinase activity. A, The full-length LeCPK1 was expressed in E. coli as a GST fusion protein and the purification of recombinant LeCPK1-L was monitored by SDS-PAGE. The crude extract (1) was separated in insoluble (2) and soluble (3) fractions by centrifugation. The fusion protein was purified from the latter by affinity chromatography on a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech, Dübendorf, Switzerland; 4, flow-through; 5, eluate). The size of the fusion protein is indicated in kD. In lanes 1–4 of the Coomassie Brilliant Blue-stained gel, the protein equivalent of 5 μL of E. coli culture is shown, whereas lane 5 corresponds to 0.8 μg of purified LeCPK1-L. B, A truncated form of LeCPK1 lacking the C-terminal calmodulin-like domain (LeCPK1-S) was expressed in E. coli and purified as in A. C, Recombinant LeCPK1-L and -S (L and S; 0.5 μg in each lane, two samples each) were separated by SDS-PAGE. Top, Coomassie Brilliant Blue-stained gel; lower, corresponding western blot. The blot was cut in two along the central lane (M) containing size markers (Bio-Rad Laboratories, Hercules, CA), and the two halves were assayed for autophosphorylation activity in absence (left) or presence (right) of Ca²⁺. The blot was autoradiographed for detection of incorporated ³²P. D, LeCPK1-L autophosphorylated in presence of [γ³²P]ATP was hydrolyzed in 6 n HCl. The hydrolysate was spiked with phospho-amino acid standards and separated by two-dimensional thin-layer chromatography (TLC). The plate was first sprayed with ninhydrin for detection of phospho-amino acid standards (left) and then autoradiographed (right).