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View additional product information for Pichia methanolica Expression System - FAQs (K178001)
8 product FAQs found
The Pichia methanolica Expression System has been discontinued. As an alternative, we offer the Pichia Pink Expression System (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/yeast-protein-expression/pichiapink-yeast-expression-systems.html)
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We recommend storing yeast frozen at -80 degrees C in 15% glycerol. Glycerol stocks are good indefinitely (unless there are numerous freeze-thaws). When making a glycerol stock, we recommend using an overnight culture and concentrating it 2-4 fold. Spin down cells and suspend in 25-50% of the original volume with glycerol/medium. It is better to store frozen cells in fresh medium plus glycerol, rather than simply adding glycerol into the overnight culture.
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We offer the original Pichia pastoris expression systems, PichiaPink expression system, and Saccharomyces cerevisiae yeast expression system for expression of recombinant proteins. Both P. pastoris and S. cerevisiae have been genetically well-characterized and are known to perform many posttranslational modifications.
The P. pastoris expression system combines the benefits of expression in E. coli (high-level expression, easy scale-up, and inexpensive growth) and the advantages of expression in a eukaryotic system (protein processing, folding, and posttranslational modifications), thus allowing high-level production of functionally active recombinant protein. As a yeast, Pichia pastoris shares the advantages of molecular and genetic manipulations with Saccharomyces cerevisiae, and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia pastoris very useful as a protein expression system. The Pichia expression vectors contain either the powerful alcohol oxidase (AOX1) promoter for high-level, tightly controlled expression, or the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for high-level, constitutive expression. Both inducible and constitutive expression constructs integrate into the P. pastoris genome, creating a stable host that generates extremely high protein expression levels, particularly when used in a fermentor. The Pichia pastoris expression systems we offer include:
- PichiaPink Yeast Expression System: Newer Pichia pastoris expression system that contains both low- and high-copy plasmid backbones, 8 secretion signal sequences, and 4 yeast strains to help optimize for the highest yield possible of the recombinant protein. All PichiaPink vectors contain the AOX1 promoter for high-level, inducible expression and the ADE2 marker for selecting transformants using ADE2 complementation (i.e., by complementation of adenine auxotrophy) rather than antibiotic selection. However, they express the ADE2 gene product from promoters of different lengths, which dictate the copy number of the integrated plasmids. The pPink-LC vector has an 82 bp promoter for the ADE2marker and offers low-copy expression, and the pPink-HC vector has a 13 bp promoter for the ADE2marker and offers high-copy expression. The system also includes the pPinkalpha-HC vector (containing S. cerevisiae alpha-mating factor pre-sequence) for high copy number secreted expression, and provides eight secretion signal sequences for optimization of secreted expression.
- EasySelect Pichia Expression Kit: One of the original Pichia expression kits that contains the pPICZ and pPICZalpha vectors, for intracellular and secreted expression, respectively, of the gene of interest. These vectors contain the AOX1 promoter for high-level, inducible expression and the Zeocin antibiotic resistance marker for direct selection of multi-copy integrants. They facilitate simple subcloning, simple purification, and rapid detection of expressed proteins.
- Original Pichia Expression Kit: The kit includes the pPIC9, pPIC3.5, pHIL-D2, and pHIL-S1 vectors, each of which carries the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. pPIC9 carries the S. cerevisiae alpha-factor secretion signal while pHIL-S1 carries the Pichia pastoris alkaline phosphatase signal sequence (PHO) to direct transport of the protein to the medium. pHIL-D2 and pPIC3.5 are designed for intracellular expression.
- Multi-Copy Pichia Expression Kit: This kit is designed to maximize expression and contains the pPIC3.5K, pPIC9K, and pAO815 vectors, which allow production and selection of Pichia strains that contain more than one copy of the gene of interest. They allow isolation and generation of multicopy inserts by in vivo methods (pPIC3.5K and pPIC9K) or in vitro methods (pAO815). All of these vectors contain the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K and pAO815 remain intracellular. The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance marker 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 the kanamycin resistance gene and the gene of interest are integrated into the genome.
- For expression in S. cerevisiae, we offer the pYES Vector Collection. Each pYES vector carries the promoter and enhancer sequences from the GAL1 gene for inducible expression. The GAL1 promoter is one of the most widely used yeast promoters because of its strong transcriptional activity upon induction with galactose. pYES vectors also carry the 2m origin and are episomally maintained in high copy numbers (10-40 copies per cell).
Yeast is a single-celled, eukaryotic organism that can grow quickly in defined media (doubling times are typically 2.5 hr in glucose-containing media) and is easier and less expensive to use for recombinant protein production than insect or mammalian cells (see table below). These positive attributes make yeast suitable for use in formats ranging from multi-well plates, shake flasks, and continuously stirred tank bioreactors to pilot plant and industrial-scale reactors.
The most commonly employed species in the laboratory are Saccharomyces cerevisiae (also known as Baker's or Brewer's yeast) and some methylotrophic yeasts of the Pichia genus. Both S. cerevisiae and P. pastoris have been genetically characterized and shown to perform the posttranslational disulphide bond formation and glycosylation that is crucial for the proper functioning of some recombinant proteins. However, it is important to note that yeast glycosylation does differ from that in mammalian cells: in S. cerevisiae, O-linked oligosaccharides contain only mannose moieties, whereas higher eukaryotic proteins have sialylated O-linked chains. Furthermore S. cerevisiae is known to hyperglycosylate N-linked sites, which can result in altered protein binding, activity, and potentially yield an altered immunogenic response in therapeutic applications. In P. pastoris, oligosaccharides are of much shorter chain length and a strain has been reported that can produce complex, terminally sialylated or “humanized” glycoproteins.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
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The use of antibiotics is not recommended because most antibiotics become inactivated at the low pH of the medium during Pichia fermentation. In other words, addition of antibiotics such as Ampicillin or Kanamycin won't hurt the fermentation process, but because of the low pH the antibiotics become inactivated or may even precipitate out. For best results, use good sterile techniques.
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Yes, Pichia should be done under BSL-1 conditions. BSL-1 is the lowest biosafety level.
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Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.