Figure 4

Signal peptide sequence, heterologous expression, and translocation of mAP. (A) N-terminal amino acid sequence of mAP showing the predicted Tat motif and signal peptide cleavage site at Ala⁸¹. (B) Expression of mAP in the E. coli BL21(DE3) transformant harboring pET-mAP at various temperatures from 15°C to 37°C. Lane M, protein standards; lane EV, total proteins expressed in BL21(DE3) cells with pET-21a at 15°C; lane T, IPTG-induced total proteins of BL21(DE3) with pET-mAP at various temperatures. (C) Secretion of mAP. mAP-expressing cells grown with 0.5 mM IPTG for 24 h were fractionated into total (lane T), periplasmic (lane P), and cytoplasmic/membrane (lane C) fractions and subjected to SDS-PAGE. Lanes M and EV are the same as in (B). (D) Purification of mAP. mAP expressed at 15°C was purified using the periplasmic fraction. Lane M, protein standards; lane P, IPTG-induced periplasmic fraction; lane mAP; purified mAP protein after dialysis and concentration. Arrows indicate precursor and mature mAP.