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Category: Expression Vectors
Initial Tab: Overview
Pichia Expression Vectors.
Recombinant protein expression in Pichia is most common from strains with expression vectors integrated into the Pichia genome. Genomic integration results in stable strains for fermentation at which Pichia excels. Expression strains can be engineered to secrete proteins into a defined medium, at multiple grams/liter - on demand. However, each gene and the related expression project goals are unique and may benefit from an alternative expression system. Factors to consider include: 1) intracellular versus secreted expression 2) what kind of promoter (inducible, constitutive, strong, weak) 3) the selection marker 4) the specific gene/ORF sequence, and 5) the location of the expression cassette in the Pichia genome.
BioGrammatics has created and tested numerous expression vectors to optimize the expression of hundreds of genes for specific applications including both intracellular and extracellular expression, the expression of membrane proteins, expression for high-level protein production, as well as the expression of proteins in metabolic pathways and proteins that support optimal processing of a recombinant protein product. Please contact BioGrammatics with inquiries on specific expression vector needs (firstname.lastname@example.org).
Following are a core set of BioGrammatics expression vectors designed for tightly regulated, strongly inducible expression with the Pichia Alcohol Oxidase I promoter (pAOX1). Even toxic genes can be expressed with the AOX1 promoter because little, if any, protein is made in Pichia prior to methanol induction. This core set includes vectors with, and without, a secretion signal (s1) and three different selectable markers (markers for G418, Noursethricin/Nat, or zeocin resistance). BioGrammatics recommends our pJAG and pJAN vectors for most initial expression tests. These vectors are available for immediate delivery.
All of BioGrammatics pJ – vectors (Jeannies vectors) are 4-5 kb plasmids with the ampicillin gene for selection in E. coli and a “seamless” cloning scheme. The cloning scheme is based on type IIS restriction enzymes that cut outside of their recognition sequence; therefore, restriction enzyme binding sites are removed in the final expression vector and an ORF can be added without unwanted codons.
|pJAN–s1||extracellular expression vector, S. cerevisiae α mating factor leader, nourseothricin selection||Details|
|pJAZ–s1||extracellular expression vector, S. cerevisiae α mating factor leader, zeocin selection||Details|
|pJAG–s1||extracellular expression vector, S. cerevisiae α mating factor leader, G418 selection||Details|
|pJAN||intracellular expression vector, nourseothricin selection||Details|
|pJAZ||intracellular expression vector, zeocin selection||Details|
|pJAG||intracellular expression vector, G418 selection||Details|