High-Level Expression in Escherichia coli of Alkaline Phosphatase from Thermus caldophilus GK24 and Purification of the Recombinant Enzyme

摘要

High-level expression of Thermus caldophilus GK24 alkaline phosphatase {Tea APase) was achieved in Escherichia coli using the pET-based expression plasmids, pEAPl and pEAP2. In the case of plasmid pEAP2, the signal peptide region of Tea APase was replaced by the PelB leader peptide of expression vector pET-22b(+). Furthermore, the expression level was somewhat higher than that of plasmid pEAPl. A rapid purification procedure of Tea APase overproduced in E. coll was developed which involved heating to denature E. coli proteins followed by HiTrap Heparin HP column chromatography. Optimal temperature and pH and Mg~(2+) dependence of the recombinant Tea APase were similar to those of native enzyme isolated from T. caldophilus GK24.