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Invitrogen™ Multi-Copy Pichia Expression Kit

Catalog No. K175001
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Catalog No. K175001 Supplier Invitrogen™ Supplier No. K175001

Description

Includes

20μg each of pPIC3.5K, pPIC9K, and pAO815 vectors, Pichia pastoris strains GS115 (his4), KM71 (arg4 his4 aox1::ARG4), control strain GS115 Albumin, control strain GS115 β-gal, Spheroplast Transformation Kit, and 2μg each 5' AOX1, 3' AOX1, and α-factor sequencing primers.

Contains pPIC3.5K, pPIC9K, and pAO815 vectors for production and selection of Pichia strains that contain more than one copy of gene of interest

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

Specifications

Content And Storage Store vectors and primers at -20°C. Store strains at room temperature. All components are guaranteed stable for 6 months when properly stored.
Protein Tag Untagged
Quantity 1 Kit
Vector pPIC

For Research Use Only. Not for use in diagnostic procedures.