High-level expression in Eschetichia coli of alkaline phosphatase from Thermus caldophilus GK24 and purification of the recombinant enzyme

High-level expression of Thermus caldophilus GK24 alkaline phosphatase (Tca APase) was achieved in Escherichia coli using the pET-based expression plasmids, pEAP1 and pEAP2. In the case of plasmid pEAP2, the signal peptide region of Tca APase was replaced by the PelB leader peptide of expression vector pET-22b(+). Furthermore, the expression level was somewhat higher than that of plasmid pEAP1. A rapid purification procedure of Tca APase overproduced in E. coli was developed which involved heating to denature E. coli proteins followed by HiTrap Heparin HP column chromatography. Optimal temperature and pH and Mg2+ dependence of the recombinant Tca APase were similar to those of native enzyme isolated from T. caldophilus GK24.