Pichia pastoris as a recombinant protein expression host: Secretion optimization of a single-chain antibody fragment and of a cancer-testis antigen

• Main Author: Damasceno, Leonardo Miranda
• Format: Dissertation
• Language: English
• Subjects: biology, microbiology
• ISBN: 0549198970; 9780549198970

Pichia pastoris has become increasingly popular as a heterologous protein expression platform. Despite its success, P. pastoris still needs to go through optimization processes if one wants to increase or obtain correct recombinant protein expression.
The first part of this work shows the optimization of a fermentation process for the production of a single-chain Fv antibody fragment (A33scFv), resulting in yields exceeding 4gL-1 of secreted product. Methanol concentration, as well as the effect of pH on A33scFv production and biomass accumulation during the induction phase were studied. A33scFv production was found to increase with higher methanol concentrations, reaching 4.3gL -1 after 72 h induction with 0.5% (v/v) methanol. Protein production was also greatly affected by pH, resulting in higher yields (4.88gL -1) at lower pH values. Purified A33scFv was also tested for functionality and showed binding to the target antigen.
In the second part of this study, further optimization of A33scFv secretion in P. pastoris was done by the co-overexpression of ER-resident chaperones. Cells were engineered to co-overexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv. Co-overexpression of BiP resulted in increased secretion levels of A33scFv by approximately 3-fold. Conversely, PDI co-overexpression had no apparent effect on A33scFv secretion. Co-overexpression of BiP and PDI did not have an expected synergistic effect on A33scFv secretion and levels remained similar to the control strain. The increase in A33scFv secretion observed during BiP overexpression is believed to be due to increased protein folding capacity in the endoplasmic reticulum (ER). Moreover, overexpression of PDI resulted in increased BiP levels during induction, indicating unfolded protein response (UPR).
The last part of this work shows how the use of engineered P. pastoris cells overexpressing PDI promotes correct processing and secretion of the cancer-testis antigen NY-ESO-1. The overexpression of PDI together with NY-ESO-1 resulted in a shift from incorrectly processed to correctly processed product in the cell culture supernatant. Conversely, co-overexpression of BiP and NY-ESO-1 did not result in secretion of correctly processed product, possibly indicating that this chaperone is being sequestered by protein aggregates in the ER and inducing the UPR.