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Bioingenium

Pichia pastoris expression

Producing proteins in Pichia pastoris

The advantages of Pichia pastoris (P. pastoris) as expression system are well know: it has the ability to perform post-translational modification like disulphide bridges, and also has the microbial capacity to growth in inexpensive media up to very high cell densities. On the other hand, P. pastoris is able to secrete the recombinant protein into the culture medium, simplifying the recovery of the protein if interest.

At Bioingenium we have a large experience in recombinant expression of proteins in P. pastoris system and 20 scientific papers published.

Benefits:

  • High yield and low productions costs for human proteins
  • Removable resistance markers genes
  • Inducible AOX1 and constitutive GAP promoter
  • Multi-copy vectors
  • Improved strains by co-expression of chaperones
  • Fermentation process development


Our feasibility studies
:

Basic

  • Gene synthesis and cloning
  • Screening of 20 Pichia clones
  • Standard culture conditions
  • Analysis by SDS-PAGE and Western Blot
  • Purification by affinity chromatography

Deliverables: All purified protein from specified culture volume (alternatively, culture supernatant or cells)


Standard

  • Gene synthesis and cloning
  • Screening of 80-95 Pichia clones
  • Screening of culture conditions to maximize productivity
  • Analysis by SDS-PAGE and Western Blot
  • Purification by affinity chromatography
Deliverables: All purified protein from specified culture volume (alternatively, culture supernatant or cells)

Optional Upgrades:
  • Evaluation of two alternatives promotors (AOX1 vs GAP)
  • Evaluation of intracellular vs extracellular expression

Premium

  • Gene synthesis and cloning
  • Screening of 500 Pichia clones
  • Screening of culture conditions to maximize productivity
  • Evaluation of two alternative promotors (AOX vs GAP)
  • Analysis by SDS-PAGE and Western Blot
  • Purification by affinity chromatography
Deliverables: All purified protein from specified culture volume (alternatively, culture supernatant or cells)

Optional Upgrades:
  • Test improved strain co-expressing chaperones
  • Evaluation of intracellular vs extracellular expression
  • Evaluation of alternatives secretion signals: ?-factor, PHO and native


Our protein production in bioreactor:

  • High cell density fermentation in bioreactor
  • Fermentation volume 5 L, 50 L and 100 L
  • Analysis by SDS-PAGE and Western Blot
  • Purification by affinity chromatography
Deliverables: All purified protein from specified culture volume (alternatively, culture supernatant or cells)


Quality certification: