Invitrogen™ Kit d’expression Pichia EasySelect™

The EasySelect™ Pichia Expression Kit provides all needed components for protein production in the yeast Pichia pastoris. The EasySelect™ Kit contains Pichia yeast strains, expression vectors, and reagents that enable transformation of Pichia cells. Selection with Zeocin™ antibiotic makes it easy to screen for high-copy number transformed variants.

Advantages of the Pichia pastoris expression system:

• Culture Pichia cells as easily as E. coli
• Obtain higher cell density than other microbial expression systems.
• Generate more protein per cell than other microbial expression systems.
• Benefit from eukaryotic protein processing, protein folding, and post-translational modifications.
• Easily scale-up from shake flasks up to industrial-scale fermentation.

A Proven Protein Expression System

Over the past 30 years, Pichia pastoris has been used by labs and industry around the world for producing hundreds of different proteins from many species including human (Ref 1, 2, 3). Pichia pastoris may be grown as easily as bacteria (E. coli) or yeast (Saccharomyces cerevisiae) yet offers many advantages over those systems (Ref 4). Pichia pastoris can grow to much higher cell densities than bacteria or other yeast. Pichia pastoris is capable of growing on methanol as a sole carbon source making scale-up to industrial levels relatively inexpensive.

The Power of the AOX1 Promoter

The EasySelect™ Pichia Expression Kit uses the promoter from the Pichia alcohol oxidase 1 (AOX1) gene to drive production of your protein. Pichia pastoris is capable of growing on methanol as its only carbon source. When grown on methanol as sole carbon source, AOX1 mRNA represents up to 5% of total mRNA and AOX1 enzyme can be up to 30% of cell weight (Ref 5).

The EasySelect™ Pichia Expression system is supplied with the pPICZ and pPICZα vectors. The pPICZ and pPICZα vectors contain a multiple cloning site (MCS) and allow the insertion of your gene of interest under control of the AOX1 promoter. The pPICZ and pPICZα vectors also contain a c-Myc tag for easy detection and a polyhistidine (6xHis) tag for easy detection and purification of your protein.

Since Pichia pastoris naturally secretes few proteins, you can use the extracellular signaling sequence in the pPICZα vectors to force secretion of your protein into the culture medium to simplify down-stream purification.

Get High-Copy Number Integration with Zeocin™ Antibiotic

The EasySelect™ Pichia Expression Kit utilizes the Zeocin™ antibiotic to select for transformed Pichia cells. pPICZ and pPICZα vectors contain the bleomycin gene from Streptoalloteichus hindustanus (Sh ble) that confers resistance to Zeocin™ antibiotic. The pPICZ and pPICZα vectors can be stably integrated into the Pichia genome. By increasing the concentration of Zeocin™ antibiotic, you can easily select for clones with the highest number of integrants and highest protein expression levels.

The EasySelect™ Pichia Expression Kit contains the following components:

• Stab vials of Pichia pastoris and E. coli strains for starting your cultures.
• Solutions for making transformation-competent Pichia cells.
• pPICZ and pPICZα vectors for fusing your gene to the AOX1 promoter.
• Sequencing primers for confirming insertion of your gene in the pPICZ vectors.
• Zeocin™ antibiotic for selecting transformed Pichia cells.

References

1. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45-66. [PubMed]

2. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol. 2002 Aug;13(4):329-32. [PubMed]

3. Cregg JM. Introduction: distinctions between Pichia pastoris and other expression systems. Methods Mol Biol. 2007;389:1-10. [PubMed]

4. Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T. Expression in the yeast Pichia pastoris. Methods Enzymol. 2009;463:169-89. [PubMed]

5. Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol. 1989 Mar;9(3):1316-23. [PubMed]