Production of recombinant protein in Pichia pastoris by fermentation

Abstract

This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes approximately 2 wk for each individual clone.

Cultivation strategies to enhance productivity of Pichia pastoris: A review

“…Conventional P. pastoris protocols describe feeding profiles for "simple" recombinant protein production without customised design of the feed profile (Invitrogen, 2002, Stratton et al, 1998, Tolner et al, 2006. What standard protocol is suitable for a particular strain depends on the strain-specific maximum specific growth rate with methanol and the optimum µ-operational range for product formation.…”

“…inducible) production of heterologous proteins (Invitrogen, 2002, Stratton et al, 1998, Tolner et al, 2006. The typical P. pastoris cultivation process, therefore, follows a threestage strategy (Fig.…”

“…From the three fedbatch protocols often used (Invitrogen, 2002, Stratton et al, 1998, Tolner et al, 2006, time courses of biomass production, and from these, specific growth rates were calculated and are shown in Fig. 6.…”

“…Available cultivation protocols are mainly based on those described in the commercial Invitrogen expression kit (Invitrogen, 2002, Stratton et al, 1998, Tolner et al, 2006. However, a recent trend is to move away from standard protocols (i.e.…”

Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants

“…So far, however, only few reports have mentioned the production of heterogeneous fusion proteins such as antibody-enzyme constructs, giving limited information on the production procedure itself [11,12]. While standard approaches and detailed procedures for the expression of recombinant proteins in Pichia have long been published [13] and recently been updated [14,15], production of a given protein, and of heterogeneous proteins in particular, may still pose a formidable challenge. Hence, seeking to improve the production of fusion proteins based on A33scFV, a single-chain antibody directed against the gpA33 antigen of colorectal cancer, we explored various production and purification strategies employing commercially available P. pastoris strains.…”