A comparative study on the secretion of alkaline phosphatase in Escherichia coli

Secretion of alkaline phosphatase (AP), a homodimeric and disulfide bond-containing protein, fused with the YfhG and the TorA signal peptides via the Sec and the Tat pathways, respectively, in Escherichia coli were investigated. The efficiency of AP translocation via the Sec pathway in MC4100, 75.5 ± 0.7%, was higher than that via the Tat pathway, 66.5 ± 0.7%. The amount of active, periplasmic AP, as suggested by the results of AP activity analysis, translocated via the Sec pathway was more than threefold that via the Tat pathway. The efficient secretion of AP via the Sec pathway alleviated protein aggregations, thus reducing the formation of alkaline phosphatase inclusion bodies in the cytoplasm. The translocation of AP in E. coli strain DR473 with relatively oxidative cytoplasm confirms the dual specificity of the YfhG signal peptide for the Sec and Tat machineries. Secretion of AP via the Tat pathway in MC4100, on the other hand, suggests that the formation of disulfide bonds may not be necessary for AP secretion via the Tat pathway. The failure of co-expressing TorD, a molecular chaperone, in enhancing the translocation of AP via the Tat pathway in DR473, and the results of in vitro on-column binding experiments suggest that proper binding to the signal peptide by TorD is a prerequisite for enhancement in protein translocation.