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. 2003 Apr 17;100(9):5022–5027. doi: 10.1073/pnas.0931263100

Figure 2.

Figure 2

Knockout of OCHI in P. pastoris. (A) The OCHI knockout plasmid pJN329 contains the P. pastoris URA3 gene flanked by LacZ repeats and 2,878 bp upstream (5′) and 1,011 bp downstream (3′) of the OCHI gene of P. pastoris. (B) Immunoblot of Och1p in Pichia wild type (JC308, lane 1) and och1 mutant (BK64-1, lane 2). The same amount of cell-free extract (50 μg per lane) was used, and Och1p was detected by using an Och1p peptide antibody followed by ECL. (C) The reporter protein K3 was expressed in Pichia wild type BK64 (lane 1) and an och1 mutant, P. pastoris BK64-1 (lane 2). K3 was purified by Ni-affinity chromatography, separated by SDS/PAGE (4–20% gradient) under reducing conditions, and visualized by silver staining. The same amount of K3 (200 ng per lane) was loaded.