FIG. 1.
Construction of recombinant plasmids pBAS01, pBAS02, and pBAS03, derived from the P. pastoris expression and secretion vector pPICZαA, containing the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s), including the nucleotides encoding the Kex2 signal cleavage site, fused in frame to the entL50A and/or entL50B structural gene and under the control of the methanol-inducible alcohol oxidase promoter (PAOX1). Plasmid sizes are given in base pairs. Only relevant restriction enzymes sites are indicated. 5′ AOX1, promoter region; AOX1 TT, transcription termination; PTEF1, transcription elongation factor 1 that drives the expression of the Sh ble gene in Pichia; PEM7, constitutive promoter driving the expression of the Sh ble gene in E. coli; Zeo gene, zeocin resistance (Sh ble gene); CYC1 TT, transcription terminator; pUC ori, maintenance and high-copy replication in E. coli; entL50A, structural gene of EntL50A; entL50B, structural gene of EntL50B; PLAC, constitutive promoter driving the expression of the lacZ gene in E. coli; f1 ori, rescue of single-stranded DNA; Kan gene, kanamycin resistance; Amp gene, ampicillin resistance.