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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.

Brand:  Invitrogen K175001

Product Code. 17804890

  • 19390.00 DKK / Each

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Description

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.
TRUSTED_SUSTAINABILITY
Specifications

Specifications

Ampicillin (AmpR), Gentamicin (GmR)
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.
Yeast Cells
AOX1
pPIC
Yeast
Expression Kit
1 Kit
KM71
Restriction Enzyme/MCS
Kit
P. pastoris
Cell-Based Expression
Protein Expression
Untagged
Ampicillin (AmpR), Gentamicin (GmR)
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For Research Use Only. Not for use in diagnostic procedures.