Multi-Copy Pichia Expression Kit

The Multi-Copy Pichia Expression Kit contains the pPIC3.5K, pPIC9K, and pAO815 vectors for production and selection of Pichia strains that contain more than one copy of the gene of interest.

Supplier:  Invitrogen K175001

Catalog No. K175001

Description

• The Multi-Copy Pichia Expression Kit contains the pPIC3.5K, pPIC9K, and pAO815 vectors for production and selection of Pichia strains that contain more than one copy of the gene of interest
• In many cases, increased copy number has led to increased expression levels
• These vectors are also available separately

pPIC9K and pPIC3.5K

• The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance gene that confers resistance to Geneticin™ Reagent in Pichia
• Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin™ Reagent
• Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to Geneticin™ Reagent
• The ability to grow in high concentrations of Geneticin™ indicates that multiple copies of the kanamycin resistance gene and the gene of interest are integrated into the genome
• The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K remain intracellular.

pAO815

• pAO815 is a Pichia expression vector designed to clone multiple copies of a gene into a single vector
• The vector contains a Bgl II site upstream of the 5´ AOX1 gene and a unique BamH I site downstream of the 3´ AOX1 transcription termination (TT) signal
• Four steps are required to generate multiple copies of the gene of interest:
  • 1. The gene is cloned into the unique EcoR I site in the vector.
  • 2. The construct is digested with BamH I and Bgl II to release the “expression cassette” containing the AOX1 promoter, gene of interest, and 3´ AOX1 TT.
  • 3. Concatemers of the expression cassette are generated by ligation in vitro.
  • 4. The multiple copies are inserted back into the pAO815 vector and transformed into Pichia.

Specifications

• Antibiotic Resistance Bacterial: Ampicillin (AmpR), Gentamicin (GmR)
• Content And Storage: The Multi-Copy Pichia Expression Kit includes 20 μg each of pPIC3.5K, pPIC9K, and pAO815 vectors, Pichia pastoris strains GS115 (his4), KM71 (arg4 his4 aox1::ARG4), control strain GS115 Albumin, control strain GS115 β-gal, Spheroplast Transformation Kit, and 2 μg each 5´ AOX1, 3´ AOX1, and α-factor sequencing primers. The pP1C9K, pPIC3.5K, and pAO815 vectors are also available separately.
  
  Store vectors and primers at -20°C. Store strains at room temperature. All components are guaranteed stable for 6 months when properly stored.
• Cell Type: Yeast Cells
• Promoter: AOX1
• Vector: pPIC
• Expression System: Yeast
• Product Type: Expression Kit
• Quantity: 1 Kit

• Bacterial or Yeast Strain: KM71
• Cloning Method: Restriction Enzyme/MCS
• Format: Kit
• Species: P. pastoris
• Expression Mechanism: Cell-Based Expression
• For Use With (Application): Protein Expression
• Protein Tag: Untagged
• Selection Agent (Eukaryotic): Ampicillin (AmpR), Gentamicin (GmR)

For Research Use Only. Not for use in diagnostic procedures.