Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: The importance of codon optimization and clone selection

Abstract

In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.

Keywords::

• Recombinant protein production
• integral membrane proteins
• P. pastoris

Acknowledgements

We thank Anna Polyakova for assistance in making the Opt-hAQP4 clone and the Carnegie Foundation.

Declaration of interest: This work was supported by contracts LSHG-CT-2004-504601 (E-MeP), LSHG-CT-2006-037793 (OptiCryst), HEALTH-F4-2007-201924 (EDICT) and QLG2-CT-2002-00988 (SPINE). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.