Figure 1

Immuno-chromogenic but not chromogenic (direct) stains specifically detect exported proteins. (Figure 1a) Chromogenic stain: Colonies derived from P.pastoris GS115 parental host strains (h) (patches 8, 9) and either strain GS115-pPIC9K Vector (h-V) (patches 12, 13) or exporting Human Serum Albumin, strain GS115-HSAsec (H) (patches 1, 2, 5, 6, 7) or parental strain GS115-EAP(EA3#27) (E(P) exporting α-sec-EAP (patches 3, 4) or the non-exporting strain GS115-β-Gal retaining intracellular β-galactosidase (β-G) (patches 10, 11) were patched onto BMGYA plates overlain with a nitrocellulose (NC) membrane. After 16 hours at 30⁰C the membrane was lifted onto a BMMYA plate and incubated for full induction at 30°C for an additional 24 hours. The NC membrane was then lifted onto Whatman 3MM Chromatography paper soaked with NBT/BCIP and monitored for differential staining of the strains. (Figure 1b) Strains H, α-sec-EAP non-expressor (translational stops in the sequence-E(D2), E(F2), α-sec-EAP expressor E(P), h-V, β-G and h were patched in replicate sets as labeled, onto the top and bottom rows of the top (Figure 1b1, b2, b3) and bottom (Figure b1’, b2’, b3’) halves of a BMGYA plate. This plate was sequentially replicated onto BMMYA plates overlain with nitrocellulose membranes (BMMYA-NC) and supplemented with (0%, 0.01%, 0.025%, 0.05%, 0.075%, 0.1%, 0.2%, 0.3%, 0.5%, 1.0% methanol), MDA plates and BMGYA plates. Representative methanol induction results of series of concentrations are shown in (1b1), (1b1^/) at 0.01% (1b2), (1b2^/) at 0.05% and (1b3), (1b3^/) at 0.2%. Upper and lower halves of nitrocellulose membrane from each plate were respectively immuno-chromogenically stained by reaction with antibodies for HSA with primary/(1°)anti - HSA/(2°)anti-Rb-IgG-AP and for EAP with primary (1⁰)anti-EAP/secondary (2°)anti-Rb-IgG-AP followed by reaction with NBT/BCIP substrate which also serves as substrate for endogenous phosphatases in simultaneous direct chromogenic staining as control for specificity.