Secreted production of an elastin-like polypeptide by Pichia pastoris

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium

Large-scale production of membrane proteins in Pichia pastoris: the production of G protein-coupled receptors as a case study.

One of the major advantages of using Pichia pastoris is that it is readily adapted to large-scale culture in bioreactors. Bioreactors allow precise regulation of cell growth parameters increasing both yields and reproducibility of the culture. P. pastoris cultures grow to very high cell densities which helps minimise culture volume and facilitates downstream processing of the sample. Here, we provide protocols for the large-scale production of the human adenosine A 2A receptor (A 2AR) and provide some details of how bioreactor cultures can be used for optimisation of expression of the human dopamine D2 receptor (D2DR). © 2012 Springer Science+business Media, LLC

Preparation of Pichia pastoris expression plasmids

When planning any heterologous expression experiment, the very first critical step is related to the design of the overall strategy, hence to the selection of the most adapted expression vector. The very flexible Pichia pastoris system offers a broad range of possibilities for the production of secreted, endogenous or membrane proteins thanks to a combination of various plasmid backbones, selection markers, promoters and fusion sequences introduced into dedicated host strains. The present chapter provides some guidelines on the choice of expression vectors and expression strategies. It also brings the reader a complete toolbox from which plasmids and fusion sequences can be picked and assembled to set up appropriate expression vectors. Finally, it provides standard starting protocols for the preparation of the selected plasmids and their use for host strain transformation.journal article2012importe