Thermo Fisher, K174001, EasySelect™ Pichia Expression Kit

EasySelect™ Pichia Expression Kit The EasySelect™PichiaExpression Kit provides all needed components for protein production in the yeastPichia pastoris. The EasySelect™ Kit containsPichiayeast strains, expression vectors, and reagents that enable transformation ofPichiacells. Selection with Zeocin™ antibiotic makes it easy to screen for high-copy number transformed variants. Advantages of thePichia pastorisexpression system:CulturePichiacells as easily asE. coliObtain 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 SystemOver the past 30 years,Pichia pastorishas 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 pastorismay be grown as easily as bacteria (E. coli) or yeast (Saccharomyces cerevisiae) yet offers many advantages over those systems(Ref 4). Pichia pastoriscan grow to much higher cell densities than bacteria or other yeast. Pichia pastorisis capable of growing on methanol as a sole carbon source making scale-up to industrial levels relatively inexpensive. The Power of the AOX1 PromoterThe EasySelect™PichiaExpression Kit uses the promoter from thePichiaalcohol oxidase 1 (AOX1) gene to drive production of your protein. Pichia pastorisis 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™PichiaExpression 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. SincePichia pastorisnaturally 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™ AntibioticThe EasySelect™PichiaExpression Kit utilizes the Zeocin™ antibiotic to select for transformedPichiacells. pPICZ and pPICZα vectors contain the bleomycin gene fromStreptoalloteichus hindustanus (Sh ble)that confers resistance to Zeocin™ antibiotic. The pPICZ and pPICZα vectors can be stably integrated into thePichiagenome. 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™PichiaExpression Kit contains the following components:Stab vials ofPichia pastorisandE. colistrains for starting your cultures. Solutions for making transformation-competentPichia 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 transformedPichiacells. References1. 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]Antibiotic Resistance Bacterial: Zeocin™ (ZeoR)
Bacterial or Yeast Strain: GS115, KM71H, X-33
Cloning Method: Restriction Enzyme⁄ MCS
Expression Mechanism: Cell-Based Expression
Expression System: Yeast
For Use With (Application): Protein Expression
Product Line: EasySelect™
Product Type: Expression Kit
Protein Tag: His Tag (6X), c-Myc Epitope Tag
Quantity: 1 Kit
Selection Agent (Eukaryotic): Zeocin
Cell Type: Yeast Cells
Format: Kit
Promoter: AOX1
Species: P. pastoris
Vector: pPIC
Unit Size: Each