Analytical Chem JoVE (Open Access) Citable · DOI

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Name: Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies
Uploaded: 2026-07-14
Duration: 5 min 30 s
Description: Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

DOI: 10.3791/58589-v

What you'll learn

✓ Set up methanol-free recombinant protein production in Pichia pastoris using de-repression promoters
✓ Monitor cultivation parameters online during glycerol-fed small-scale bioreactor experiments
✓ Analyze and quantify de-repressed protein expression results from PDC and PDF promoter systems

Protocol

Biopharma Insights This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

Difficulty

advanced

Total time

~3–5 days (culture growth and harvesting; protein expression analysis adds 1–2 days)

Model organism

Pichia pastoris (Komagataella phaffii)

Biosafety

BSL-1

Steps

Cultivate and harvest Pichia pastoris main culture

Grow Pichia pastoris cells in controlled bioreactor conditions with online monitoring of cultivation parameters. Apply constant glycerol feed to regulate carbon source and induce de-repressed protein expression via PDC and PDF promoters.

▶ 00:43

Analyze de-repressed protein expression results

Quantify and evaluate recombinant protein production from cultures using PDC and PDF carbon source regulated promoters. Compare expression levels and characterize protein yield from methanol-free induction.

▶ 03:30

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