Thermo Fisher, K175001, Multi-Copy <i>Pichia</i> Expression Kit

Quantity: 1 Kit
Unit Size: Each
• 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
• These vectors are also available separatelypPIC9K and pPIC3.5K
• The 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
• The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K remain intracellular.pAO815
• pAO815 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.