Journal: Journal of Fungi

Article Title: Two-Step Engineering of Food-Grade Aspergillus oryzae via Endogenous Signal Peptides and Vesicle Trafficking Proteins to Enhance Carrier-Free Protein Secretion

doi: 10.3390/jof12040289

Figure Lengend Snippet: Analysis of Aoκ-casein production in recombinant A. oryzae . ( A ) Protein separation by SDS-PAGE. ( B ) Western blot analysis using a bovine κ-casein polyclonal antibody. Lane bovine κ-casein: 1 µg of κ-casein standard (~25 kDa); lane M: prestained protein molecular weight marker; Lanes 1–4 cell: intracellular protein extracts from the wild type, Aoκ-casein, SPAoalp1-Aoκ-casein, and SPAoalp1-Aoκ-casein + Aosly1 strains, respectively; lanes 1–4 sup: corresponding extracellular protein extracts from the same strains. The black arrow indicates the κ-casein standard, and red arrowheads indicate Aoκ-casein produced by recombinant strains. Each lane was loaded with 50 µg of intracellular protein or 20 µg of extracellular protein. Abbreviations: Aoκ-casein, A. oryzae –derived κ-casein; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Article Snippet: After washing and blocking with BlockProTM (Visual Protein, Taipei, Taiwan), the wells were incubated with bovine κ-casein polyclonal antibody (Bioss Inc., Woburn, MA, USA; 1:3500 dilution).

Techniques: Recombinant, SDS Page, Western Blot, Molecular Weight, Marker, Produced, Derivative Assay, Polyacrylamide Gel Electrophoresis

Journal: Journal of Fungi

Article Title: Two-Step Engineering of Food-Grade Aspergillus oryzae via Endogenous Signal Peptides and Vesicle Trafficking Proteins to Enhance Carrier-Free Protein Secretion

doi: 10.3390/jof12040289

Figure Lengend Snippet: Effect of yeast extract (YE) supplementation on biomass formation and recombinant Aoκ-casein production in A. oryzae . ( A ) Biomass accumulation and total extracellular protein concentration in cultures grown with increasing YE concentrations. ( B ) Aoκ-casein titers quantified by ELISA using a bovine κ-casein polyclonal antibody. Values are presented as the mean ± standard deviation ( n = 3 biological replicates). Bars labeled with different lowercase letters (a–d) indicate statistically distinct groups based on one-way ANOVA followed by DMRT ( p < 0.01). Abbreviations: DW, dry weight; Aoκ-casein, A. oryzae -derived κ-casein; ELISA, enzyme-linked immunosorbent assay; DMRT, Duncan’s multiple range test.

Article Snippet: After washing and blocking with BlockProTM (Visual Protein, Taipei, Taiwan), the wells were incubated with bovine κ-casein polyclonal antibody (Bioss Inc., Woburn, MA, USA; 1:3500 dilution).

Techniques: Recombinant, Protein Concentration, Enzyme-linked Immunosorbent Assay, Standard Deviation, Labeling, Derivative Assay

Journal: Journal of Fungi

Article Title: Two-Step Engineering of Food-Grade Aspergillus oryzae via Endogenous Signal Peptides and Vesicle Trafficking Proteins to Enhance Carrier-Free Protein Secretion

doi: 10.3390/jof12040289

Figure Lengend Snippet: Summary of the workflow schematic of the two-step engineered targets for improving carrier-free protein secretion with integrative outcomes. Step 1 is quantified as a fold increase relative to the no-SP control and Step 2 as the additional fold increase relative to the SP-only construct. Validation of the engineered secretory system was complemented through extracellular bovine Aoκ-casein and supernatant from AoPAFB-expressing strain. Abbreviations: GFP, green fluorescent protein; SP, signal peptide; VTPs, vesicle trafficking proteins; Aoκ-casein, A. oryzae -derived κ-casein; PAFB, Penicillium chrysogenum antifungal protein B; MIC 90 , Minimum inhibitory concentration required to inhibit 90% of growth.

Article Snippet: After washing and blocking with BlockProTM (Visual Protein, Taipei, Taiwan), the wells were incubated with bovine κ-casein polyclonal antibody (Bioss Inc., Woburn, MA, USA; 1:3500 dilution).

Techniques: Control, Construct, Biomarker Discovery, Expressing, Derivative Assay, Concentration Assay