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Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures.

Microbial cell factories (2008-10-14)
Shweta Singh, Adrien Gras, Cédric Fiez-Vandal, Jonathan Ruprecht, Rohini Rana, Magdalena Martinez, Philip G Strange, Renaud Wagner, Bernadette Byrne
ABSTRACT

The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures. Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A2AR, and therefore more suitable for further functional and structural studies. Large-scale expression of the A2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Histidine, ReagentPlus®, ≥99% (TLC)
Sigma-Aldrich
L-Histidine, suitable for cell culture, meets EP, USP testing specifications, from non-animal source
SAFC
L-Histidine, PharmaGrade, Ajinomoto, EP, USP, manufactured under appropriate GMP controls for Pharma or Biopharmaceutical production, suitable for cell culture
Sigma-Aldrich
L-Histidine, BioUltra, ≥99.5% (NT)
Supelco
L-Histidine, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
L-Histidine, certified reference material, TraceCERT®

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