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. 2006 Feb;72(2):986–993. doi: 10.1128/AEM.72.2.986-993.2006

FIG. 4.

FIG. 4.

Pichia expression of XYL-6H. (A) XYL-6-transformed Pichia clones (clones 141 and 173), a positive control-expressing M. grisea xylanase XYL-4 (GenBank no. AY144349; manuscript in preparation), and a clone carrying the empty vector pPic3.5K were induced for protein expression in 1 liter of induction medium for 80 h. Specific endo-β-1,4-xylanase activity in the culture filtrate was assayed with 100 mM phosphate, pH 6.0, using RBB-xylan (Sigma) as the substrate according to Wu et al. (37). Total protein content was measured using Protein Assay Reagent purchased from Bio-Rad Laboratories (Hercules, CA). (B) SDS-PAGE and Western blot analysis of secreted proteins in the culture filtrate (30 μl). Lane M, molecular mass markers; lane P, 0.5 μg of HisLink-purified XYL-6H from the culture filtrate of Pichia clone 173; lane W, Western blot analysis of lane P using an alkaline phosphatase-conjugated anti-myc antibody.