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

Abstract

Background: Human BiP, or GRP78, is a molecular chaperone mainly found in the endoplasmic reticulum (ER). However, a growing amount of data also associates BiP with many distinct functions in subcellular locations outside the ER. Notably, several diseases have been BiP-related, so the protein could potentially be used for therapeutic purposes. This study aimed to optimize a high cell-density fermentation process for the production of recombinant human BiP (rhBiP) in yeast Pichia pastoris in a mineral medium.

Results: P. pastoris cells successfully synthesized and secreted full-length rhBiP protein in a complex growth medium. However, secreted rhBiP titer was considerably lower when P. pastoris was cultivated in a defined mineral basal salt medium (BSM). During rhBiP synthesis optimization in shake flasks, it was found that the addition of reducing compounds (DTT or TCEP) to mineral BSM medium is essential for high-yield rhBiP production. Furthermore, rhBiP secretion in the BSM medium was significantly increased by feeding yeast with an additional carbon source. The addition of 2 mM DTT and 0.5-1.0% of glucose/glycerol to the BSM medium increased rhBiP titer ~ 8 times in the shake flasks. Glucose/methanol mixture feeding with added 2 mM DTT before induction was applied in high-density P. pastoris fermentation in bioreactor. Oxygen-limited fermentation strategy allowed to achieve ~ 70 mg/L rhBiP in BSM medium. Hydrophobic interaction and anion exchange chromatography were used for rhBiP protein purification. 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 has ATPase activity. In addition, we show that P. pastoris-derived rhBiP effectively inhibits 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 a 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.

Keywords: Pichia pastoris; BiP; DTT; Fermentation; Mineral medium; Mixed feeding; Secretion.

Fig. 1

SDS-PAGE analysis of rhBiP secretion into different growth media in P. pastoris. Lanes were loaded with samples of rhBiP-producing clone ppBiP10 cultivated in the shake flasks with indicated culture media: YEPM– complex media; BSM– basal salt medium; BSM + YE + PE– basal salt media supplemented with yeast extract and soy peptone; BSM + YE– basal salt media supplemented with yeast extract; BSM + PE– basal salt media supplemented with soy peptone. C lane– YEPM culture media from P. pastoris transformed with control pPIC3.5 K vector. All culture growth media were concentrated 10x and 7.5 µl (obtained from 75 µl of culture medium) was loaded onto the lane. M lane – unstained protein ladder (ThermoScientific, cat. no. 26614)

Fig. 2

Selection of optimal culture medium for rhBiP secretion in shake flask cultures of P. pastoris. SDS-PAGE was performed with following culture media samples on lanes: C- P. pastoris transformed with control pPIC3.5K vector in complex YEPM culture media; rhBiP-producing clone ppBiP10 cultivated in BSM – basal salt medium; BSM + DTT – basal salt medium supplemented with 2mM DTT; BSM + TCEP - basal salt medium supplemented with 2 mM TCEP; BSM + Gluc - basal salt medium supplemented with 0,75% (m/V) glucose; BSM + DTT + Gucose - basal salt medium supplemented with 2 mM DTT and 0.75% (m/V) glucose. All cultures (YEPM and BSM media with or without additives) contained the same 0.5% amount of the inducer methanol, as described in Methods. All culture growth media were concentrated 10x, and 7.5 µl (obtained from 75 µl of culture medium) was loaded onto the lane. M lane – unstained protein ladder (ThermoScientific, cat. no. 26614)

Fig. 3

Secretion of rhBiP in high-cell-density fermentation of P. pastoris in a bioreactor. (A) SDS-PAGE analysis of culture supernatant during fermentation. 35 µl of P. pastoris clone ppBiP10 culture media, taken at different time points, was loaded onto each lane. (B) Time-course profile of rhBiP secretion in high-density P. pastoris culture during fermentation. Circles represent wet cell weight (WCW), and triangles show the concentration of the secreted rhBiP in the culture medium. 0–24 h indicates glycerol batch phase, 24–25 h - glycerol fed-batch phase, 25–28 h - transition phase, and 28–100 h - methanol/glucose fed-batch phase. Only the amount of intact secreted rhBiP protein form was calculated

Fig. 4

SDS-PAGE analysis of rhBiP purified from P. pastoris culture medium. 5 µg of purified rhBiP was loaded on the gel. M - prestained molecular ladder (ThermoScientific, cat. no. 26618)

Fig. 5

ESI-MS and N-terminal sequencing of rhBiP from P. pastoris

Fig. 6

ATPase activity of P. pastoris-secreted human BiP. The amount of released phosphate by 1 µg of either bacterial or yeast-derived rhBiP was determined after incubation at 25 °C for 75 min. with a non-radioactive procedure. Values are the mean of three separate experiments with the SD error bar

Fig. 7

Monomerization of rhBiP after exposure to ATP. Purified rhBiP derived from P. pastoris culture medium was incubated with 5mM of ATP for 15 min at 30 °C. 5 µg of protein was loaded on gel and analyzed by native PAGE. Commercial BSA was used as a control and molecular weight marker

Fig. 8

Inhibition of aggregation of Aβ₄₂ (A) and α-Syn (B) by rhBiP derived from P. pastoris. (A) Aggregation kinetics of the Aβ₄₂ peptide in solution at different rhBiP concentrations. (B) Aggregation kinetics of the alpha-synuclein in solution at presence of 5 µM rhBiP or without chaperone. All aggregation measurements were carried out using the ThT assay as described in Methods