Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Abstract

Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of CAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by displaying it on the surface of filamentous bacteriophage fd. Phosphorylationin 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-rner peptide library phosphorylated by cAPK was selected with ferric (Fe(3+)) chelation affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Theirin vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typical (R)RXS/T sequence motif. It suggested that the new method of using ferric (Fe(3+)) chelation affinity chromamraphy to identify the substrate specificity of protein kinase from random peptide library was feasible.