Optimization of human chaperone BiP production in yeast Pichia pastoris

Abstract Background Human BiP, also known as GRP78, is a molecular chaperone mainly located in the endoplasmic reticulum (ER). However, a growing amount of data also associates BiP with many different functions in subcellular locations outside the ER. Importantly, several diseases have been BiP-related and therefore it could potentially be used for therapeutic purposes. The aim of this study was to optimize a high cell density fermentation process for production of recombinant human BiP (rhBiP) in yeast Pichia pastoris in mineral medium. Results P. pastoris cells successfully synthesized and secreted full length rhBiP protein in complex growth medium. However, secreted rhBiP titer was considerably lower when P. pastoris was cultivated in defined mineral basal salt medium (BSM). During rhBiP synthesis optimization in shake flasks it was found that addition of reducing compounds (DTT or TCEP) to mineral BSM medium is essential for high yield rhBiP production. Furthermore, rhBiP secretion in BSM medium was significantly increased by feeding yeast with an additional carbon source. Addition of 2 mM DTT and 0.5-1.0 % of glucose/glycerol to BSM medium increased rhBiP titer ~ 8 times in the shake flasks. Glucose/methanol mixture feeding with 2 mM DTT addition before induction was applied in high density P. pastoris fermentation in bioreactor. Oxygen limited fermentation strategy allowed to achieve ~70 mg/L rhBiP titer in BSM medium. For rhBiP protein purification hydrophobic interaction and anion exchange chromatography were used. Approximately 45 mg rhBiP was purified from 1 L growth medium and according to SDS-PAGE ~90 % purity was reached. According to data presented in this study, rhBiP protein derived from P. pastoris is a full-length polypeptide that possesses ATPase activity. In addition, we show that P. pastoris-derived rhBiP effectively inhibited neurodegenerative disease-related amyloid beta 1-42 (Aβ42) peptide and alpha-synuclein (α-Syn) protein aggregation in vitro. Conclusions A scalable bioprocess to produce rhBiP in P. pastoris was developed providing high yield of biologically active protein in a chemically defined mineral medium. It opens a source of rhBiP to accelerate further therapeutic applications of this important protein.