Multi-Copy Pichia Expression Kit

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.