pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins

The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli P hoA/S gene that encodes an alkaline phosphatase with increased specific act ivity. The restriction sites incorporated into pSKAP/S allowed the scFv gen es to be easily transferred from pUC119-derived phagemid vectors that are u sed frequently in phage display antibody library technology. Strong transcr iptional control of expression was achieved using the tetracycline promoter , and induction of different individual clones with anhydrotetracycline res ulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion prote ins that were retained in the periplasm, these proteins could be isolated w ith a simple extraction procedure. Increased amounts of a scFv-alkaline pho sphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testin g for binding of the scFv-alkaline phosphatase fusion proteins to antigen w as possible in an ELISA without the need for additional enzyme-conjugated a ntibodies. The pSKAP/S vector was successfully used to obtain scFv fragment s from a preparation of phage-antibody clones after subcloning and expressi on of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones land clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. There fore, the pSKAP/S vector was shown to be useful in extending the range of s cFv obtained from phage display libraries. (C) 1999 Academic Press.