Thermo Fisher, K175001, Multi-Copy Pichia Expression Kit

Thermo Fisher, K175001, Multi-Copy Pichia Expression Kit The Multi-CopyPichiaExpression Kit contains the pPIC3.
5K, pPIC9K, and pAO815 vectors for production and selection ofPichiastrains that contain more than one copy of the gene of interest. In many cases, increased copy number has led to increased expression levels.
pPIC9K and pPIC3.
5KThe pPIC9K and pPIC3.
5K vectors carry the kanamycin resistance gene that confers resistance to Geneticin™ Reagent inPichia. Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin™ Reagent.
Pichiatransformants 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 (1). The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.
5K remain intracellular.
pAO815pAO815 is aPichiaexpression vector designed to clone multiple copies of a gene into a single vector. The vector contains aBglII site upstream of the 5´AOX1gene and a uniqueBamH I site downstream of the 3´AOX1transcription termination (TT) signal. Four steps are required to generate multiple copies of the gene of interest:1. The gene is cloned into the uniqueEcoR I site in the vector.
2. The construct is digested withBamH I andBglII to release the “expression cassette” containing theAOX1promoter, gene of interest, and 3´AOX1TT.
3. Concatemers of the expression cassette are generated by ligationin vitro.
4. The multiple copies are inserted back into the pAO815 vector and transformed intoPichia.
These vectors are also available separately.
Antibiotic Resistance Bacterial: Ampicillin (AmpR), Gentamicin (GmR)
Bacterial or Yeast Strain: KM71
Cloning Method: Restriction Enzyme/MCS
Expression Mechanism: Cell-Based Expression
Expression System: Yeast
For Use With (Application): Protein Expression
Product Type: Expression Kit
Protein Tag: Untagged
Quantity: 1 Kit
Selection Agent (Eukaryotic): Ampicillin (AmpR), Gentamicin (GmR)
Cell Type: Yeast Cells
Format: Kit
Promoter: AOX1
Species: P. pastoris
Vector: pPIC
Unit Size: Each