Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules

摘要

Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters Km and kcat were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a Km of 11.2 µM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 µM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface.