Development of an efficient expression system for human chaperone BiP in Pichia pastoris: production optimization and...

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Heat shock protein BiP (GRP78) is a molecular chaperone primarily in the endoplasmic reticulum.
• BiP is implicated in various diseases and shows therapeutic potential.
• This study focuses on producing recombinant human BiP (rhBiP) using Pichia pastoris.

Purpose of the Study:

• To optimize a high cell-density fermentation process for rhBiP production in Pichia pastoris.
• To develop a scalable and cost-effective method for obtaining biologically active rhBiP.
• To investigate conditions for enhancing rhBiP secretion in a mineral medium.

Main Methods:

• Utilized Pichia pastoris for recombinant protein expression.
• Optimized fermentation conditions in shake flasks and bioreactors.
• Employed hydrophobic interaction and anion exchange chromatography for purification.
• Assessed rhBiP activity and its effect on protein aggregation in vitro.

Main Results:

• Achieved high-yield rhBiP production in a defined mineral basal salt medium (BSM) with optimized conditions.
• Identified essential additives (DTT, carbon source) for increased rhBiP secretion.
• Reached approximately 70 mg/L rhBiP in bioreactor fermentation with an oxygen-limited strategy.
• Purified rhBiP to ~90% purity, demonstrating ATPase activity and in vitro inhibition of amyloid-beta and alpha-synuclein aggregation.

Conclusions:

• Developed a scalable bioprocess for producing high-yield, biologically active rhBiP in P. pastoris.
• The process utilizes a chemically defined mineral medium, facilitating further therapeutic applications.
• This work provides a reliable source of rhBiP for accelerating disease-related research.