Improving recombinant human adenosine A_2A receptor production in yeast

Abstract

Over 50% of clinically-marketed drugs target membrane proteins; in particular G protein-coupled receptors (GPCRs). GPCRs are vital to living cells, performing an active role in many processes, making them integral to drug development. In nature, GPCRs are not sufficiently abundant for research and their structural integrity is often lost during extraction from cell membranes.
The objectives of this thesis were to increase recombinant yield of the GPCR, human adenosine A_2A receptor (hA_2AR) by investigating bioprocess conditions in large-scale Pichia pastoris and small-scale Saccharomyces cerevisiae cultivations. Extraction of hA_2AR from membranes using novel polymers was also investigated.
An increased yield of hA_2AR from P. pastoris was achieved by investigating the methanol feeding regime. Slow, exponential feed during induction (μ_low) was compared to a faster, exponential feed (μ_high) in 35 L pilot-scale bioreactors. Overall hA_2AR yields were increased for the μlow cultivation (536.4pmol g^-1) compared to the μ_high148.1 pmol g^-1. hA_2AR levels were maintained in cytotoxic methanol conditions and unexpectedly, pre-induction levels of hA_2AR were detected. Small-scale bioreactor work showed that Design of Experiments (DoE) could be applied to screen for bioprocess conditions to give optimal hA2AR yields. Optimal conditions were retrieved for S. cerevisiae using a d-optimal screen and response surface methodology. The conditions were 22°C, pH 6.0, 30% DO without dimethyl sulphoxide. A polynomial equation was generated to predict hA_2AR yields if conditions varied.
Regarding the extraction, poly (maleic anhydride-styrene) or PMAS was successful in solubilising hA_2AR from P. pastoris membranes compared with dodcecyl-β-D-maltoside (DDM) detergent. Variants of PMAS worked well as solubilising agents with either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or cholesteryl hemisuccinate (CHS). Moreover, esterification of PMAS improved solubilisation, suggesting that increased hydrophobicity stabilises hA_2AR during extraction.
Overall, hA_2AR yields were improved in both, P. pastoris and S. cerevisiae and the use of novel polymers for efficient extraction was achieved.