gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
P. pastoris Yeast Recombinant Protein Expression Vector
Our Pichia pastoris recombinant protein vector system is a powerful and efficient system for expressing recombinant proteins in this yeast species. P. pastoris is methylotrophic yeast widely used for protein production for research and industrial applications.
In this vector system, the gene of interest is typically cloned under the control of either the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the alcohol oxidase 1 (AOX1) promoter, depending on whether constitutive or inducible expression is desired. The GAP promoter is a strong, constitutive promoter, while the AOX1 promoter is tightly regulated, and strongly inducible by methanol. Both promoters are capable of driving very high-level gene expression, often with the protein of interest accumulating to more than 30% of total cellular soluble protein. In general, the GAP promoter is slightly stronger than the AOX1 promoter in its induced state. We recommend that you perform a time course experiment to optimize expression of your recombinant protein and to determine the best promoter to drive its expression.
Unlike S. cerevisiae, P. pastoris metabolizes methanol as its sole carbon source (a.k.a. methylotrophic), and achieves much higher levels of recombinant protein expression than S. cerevisiae (often 10- to 100-fold higher). In addition, many techniques commonly used with S. cerevisiae can be employed with P. pastoris (e.g. complementation), and common gene annotations and genetic nomenclature simplify research using both species.
For further information about this vector system, please refer to the papers below.
|Gene. 186:37 (1997)||Description of GAP promoter|
|Mol Cell Biol. 5:1111 (1985)
Yeast. 5:167 (1989)
|Description of AOX1 promoter|
This vector system is designed for either constitutive or inducible expression of recombinant proteins in P. pastoris. The gene of interest is cloned into the vector under the control of either the constitutive GAP promoter or the methanol-inducible AOX1 promoter.
Strong expression: Both GAP and AOX1 promoters allow for very high-level expression of genes of interest.
Tightly controlled expression: If the AOX1 promoter is used, the expression of the gene of interest is generally very strongly repressed in the absence of methanol and is strongly activated when methanol is present.
Potential unwanted/leaky expression: The GAP promoter is constitutively active, so expression of the gene of interest is unregulated. Additionally, although the AOX1 promoter is generally very tightly repressed in the absence of methanol, in some cases leaky expression of the gene of interest may be a concern.
Promoter: Drives high-level transcription of the gene of interest.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
AOX1 TT: The transcription terminator from P. pastoris alcohol oxidase 1. This facilitates transcriptional termination and polyadenylation of mRNA in yeast.
Zeocin: Zeocin resistance gene. It allows the plasmid to be maintained by Zeocin selection in E. coli and yeast.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.