Multi-copy Pichia pastoris expression kit contains pPIC3.5K, pPIC9K and pAO815 vectors. For the production and selection of Pichia pastoris strains containing more than one copy of the target gene. In many cases, increased copy number results in increased expression levels.

pPIC9K and pPIC3.5K
The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance gene, making Pichia pastoris resistant to Geneticin™ Reagents develop resistance. The natural occurrence of multiple insertion events can be identified by the development of resistance to elevated levels of Geneticin™ reagent. Pichia pastoris transformants were selected on histidine-deficient medium and screened for resistance levels to Geneticin™ reagent. The ability to grow in high concentrations of Geneticin™ indicates that multiple copies of the kanamycin resistance gene and target gene have been integrated into the genome (1). The pPIC9K vector directs secretion of the expressed protein, while the protein expressed by pPIC3.5K remains within the cell.

pAO815
pAO815 is a Pichia pastoris expression vector designed for cloning multiple copies of a gene into a single vector. The vector contains a Bgl II site upstream of the 5´AOX1 gene and a uniqueBa downstream of the 3´AOX1 transcription termination (TT) signal. mH I site. Four steps are required to generate multiple copies of a target gene:
1. Clone the gene into a uniqueEcoR I site in the vector.
2. Use BamH I and Bgl II to digest the construct to release the AOX1 promoter, target gene and 3´< The “expression cassette” of i>AOX1TT.
3. Generate the concatemer of the expression cassette through in vitro ligation.
4. Re-insert multiple copies into the pAO815 vector and transform into Pichia pastoris.

These carriers are also available for purchase individually.