SELECTION OF SUBSTRATE RECOGNITION SEQUENCE OF PROTEIN-KINASE WITH FERRIC CHELATION AFFINITY-CHROMATOGRAPHY

Protein kinase substrate phage (PKS phage) was constructed by fusing t he substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by display ing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku , which was consistent with the molecular weight of the PKS-g3p fusion protein, Some weakly phosphorylated bands for both PKS phage and wild -type phage were also observed. Phage display random 15-mer peptide li brary phosphorylated by cAPK was selected with ferric (Fe3+) chelation affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. T he results showed thar 5 of 14 sequenced phages displayed the cAPK rec ognition sequence motif (R)RXS/T. Their in vitro phosphorylation analy ses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typical (R)RXS/T sequence motif. It s uggested that the new method of using ferric (Fe3+) chelation affinity chromatography to identify the substrate specificity of protein kinas e from random peptide library was feasible.