Fig 1: Genetic and phenotypic characterization of recombinant P. pastoris. (A) antibiotic selection of recombinant strains. 4 colonies of P. pastoris X-33 harboring pGAPZB-L1P18 (red circled) were used for yeast colony PCR. (B) Yeast colony PCR. Lane 1. X-33 harboring the pGAPZB without DNA insert (negative control); lane 2, plasmid pGAPZB-L1P18 (positive control); lane 3. Molecular weight marker (Invitrogen 1kb plus DNA ladder, 1650 bp); lanes 4–7: Recombinant yeast strains containing the pGAPZB-L1P18 carrying the DNA coding sequence L1P18 (1563 bp). (C) Western blot analysis of L1 protein expression in cell lysates of four recombinant clones of P. pastoris X-33 harboring pGAPZB-L1P18, probed with the anti-L1 mAb [CamVir 1] (Abcam, Cambridge, UK). Lane 1. Molecular weight markers (50 kDa and 60 kDa); lane 2, Recombinant P. pastoris X33 harboring the pGAPZB without heterologous DNA insert (negative control); lane 3, abcam HPV16 L1 protein 75 ng (positive control, 56 kDa); lanes 4–7, 4 recombinant P. pastoris X-33 colonies harboring the pGAPZB-L1P18 expressing the L1P18 protein. (D) Dot blot analysis of P18 peptide expression in cell lysates of two recombinant clones of P. pastoris X-33 harboring pGAPZB-L1P18, probed with mAb to HIV-1 V3 loop (reference: EVA3012, Center for AIDS reagents, NIBSC). Dot 1, 2: protein extracted from recombinant BCG:HIVA 222 strain expressing HIVA immunogen that contains P18 peptide (4, and 8 µL) (positive control); dot 3 and 4: protein extracted from recombinant BCG:HIVA 223 strain (4 and 8 µL) (positive control); dot 5, 6 and 7: cell lysate of recombinant P. pastoris clone#3 (4, 8 and 12 µL); dot 8, PBS (negative control); dot 9, Abcam recombinant HPV16 L1 protein (ab119880) (50 ng) (negative control); dot 10, Abcam recombinant HPV16 L1 protein (ab119880) (20 ng) (negative control); dot 11, Abcam recombinant HPV16 L1 protein (ab119880) (10 ng) (negative control); dot 12, 13 and 14: cell lysate of recombinant P. pastoris clone #4 (4, 8 and 12 µL).