Invitrogen™ Multi-Copy Pichia Expression Kit 

Description

In many cases, increased copy number has led to increased expression levels.

• pPIC9K and pPIC3.5K vectors carry 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 kanamycin resistance gene and gene of interest are integrated into genome
• pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K remain intracellular
• pAO815 is Pichia expression vector designed to clone multiple copies of a gene into single vector
• Vector contains a Bgl II site upstream of 5' AOX1 gene and unique BamH I site downstream of 3' AOX1 transcription termination (TT) signal
• Four steps are required to generate multiple copies of gene of interest: 1. The gene is cloned into unique EcoR I site in the vector
• Construct is digested with BamH I and Bgl II to release expression cassette containing AOX1 promoter, gene of interest, and 3' AOX1 TT
• Concatemers of expression cassette are generated by ligation in vitro; 4. The multiple copies are inserted back into pAO815 vector and transformed into Pichi

Pichia pastoris Expression Systems, Protein Expression, Proteins, Expression, Isolation and Analysis, Yeast Expression