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.

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 (1). 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.

These vectors are also available separately.