What is the codon usage for Pichia?

Product FAQ

Answer

It is doubtful as to whether codon usage plays as great a role in general, as is commonly believed. Translation initiation is probably more of a rate-limiting step than elongation.
Use the following codon usage list to design your gene in the order of preference:

Glycine: GGT or GGA
Glutamic acid: GAG or GAA
Aspartic acid: GAC or GAT
Valine: GTT or GTC
Alanine: GCT or GCC
Arginine: AGA or CGT
Serine: TCT or TCC
Lysine: AAG
Asparagine: AAC
Methionine: ATG
Isoleucine: ATT or ATC
Threonine: ACT or ACC
Tryptophan: TGG
Cysteine: TGT
Tyrosine: TAC
Leucine: TTG or CTG
Phenylalanine: TTC
Glutamine: CAA or CAG
Histidine: CAC or CAT
Proline: CCA or CCT

Answer Id: E9492

Was this answer helpful?

Yes
No
Thank you for your response

How can I freeze Pichia?

Product FAQ

Answer

Store glycerol stocks frozen at -80° 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.

Answer Id: E4227

Was this answer helpful?

Yes
No
Thank you for your response

My transformation is not working. Do you have any suggestions?

Product FAQ

Answer

Here are some suggestinos:

- Make sure that you have harvested cells during log-phase growth (OD <1.0 generally).
- If electroporation is being used, see the electroporator manual for suggested conditions. Vary electroporation parameters if necessary.
- Use more DNA.
- Use freshly made competent cells.
- If the LiCl transformation method is being used, try boiling the carrier DNA.

Answer Id: E9561

Was this answer helpful?

Yes
No
Thank you for your response

Will a mammalian secretion signal work in Pichia?

Product FAQ

Answer

Although the efficiency may differ from one signal to the next, in general mammalian secretion signals are functional in yeast.

Answer Id: E9494

Was this answer helpful?

Yes
No
Thank you for your response

Can YPD be used instead of BMGY-type media for pichia fermentation?

Product FAQ

Answer

Yes. The cells will do fine in YPD but there are two drawbacks: The foaming that occurs in the richer YPD is very difficult to control. The richer media makes it difficult to purify secreted proteins from the media. The BMGY formulation remedies both of these problems.

Answer Id: E3755

Was this answer helpful?

Yes
No
Thank you for your response

What are the advantages of the PichiaPink™ Yeast Expression System over the EasySelect™ Yeast Expression system?

Product FAQ

Answer

PichiaPink™ Yeast Expression System offers significant advantages compared to the original EasySelect™ Pichia system. Please see the advantages below:

- Both high and low copy enables optimization of toxic protein expression
- 8 secretion signal leader sequences
- 4 strains
- 3 protease-deficienct host strains
- Relies on adenine selection instead of an antibiotic resistance marker

Answer Id: E9481

Was this answer helpful?

Yes
No
Thank you for your response

When selecting for blasticidin-resistant transformants in the X-33 strain using pPIC6/pPIC6alpha vectors, why do I get large and small colonies on YPD plates containing 300 μg/ml blasticidin?

Product FAQ

Answer

Generally, large colonies represent transformants containing pPIC6/pPIC6alpha integrants, while small colonies represent transformants containing pPIC6/pPIC6alpha non-integrants. These non-integrants have transduced the pPIC6/pPIC6alpha plasmid, and therefore, exhibit a low level of blasticidin resistance in the initial selection process. Upon subsequent screening, these non-integrant transformants do not retain blasticidin resistance.

When choosing a blasticidin-resistant transformant for your expression studies, we recommend that you pick blasticidin-resistant colonies from the initial transformation plate and streak them on a second YPD plate containing the appropriate concentration of blasticidin. Select transformants that remain blasticidin-resistant for further studies.

Answer Id: E9562

Was this answer helpful?

Yes
No
Thank you for your response

How is the alpha factor secretion signal sequence processed?

Product FAQ

Answer

The alpha “signal sequence” (which really contains both the alpha signal sequence and pro-hormone leader sequences) is cleaved 4 times by 3 different enzymes in the Pichia cell. First, near the N-terminus by signal peptidase; second, by Kex2p after the dibasic (Lys-Arg) signal slightly upstream of the multiple cloning site, and then twice by Ste13p to remove the 2 Glu-Ala repeats.

Answer Id: E9495

Was this answer helpful?

Yes
No
Thank you for your response

Why would I pick a yeast expression system for expression of my protein, as opposed to expression systems in other hosts?

Product FAQ

Answer

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.

Answer Id: E9477

Was this answer helpful?

Yes
No
Thank you for your response

Do you need to add sulfuric acid to the Fermentation PTM Trace salts?

Product FAQ

Answer

You don't have to add sulfuric acid to your PTM1 salts or fermentation media. It would serve no purpose, other than may be help dissolve the salts.

Answer Id: E3756

Was this answer helpful?

Yes
No
Thank you for your response

What is the purpose of including sorbitol in the YPD plates used for plating Pichia cells after electroporation?

Product FAQ

Answer

Inclusion of 1 M sorbitol in YPD plates stabilizes electroporated cells, as they appear to be somewhat osmotically sensitive.

Answer Id: E9531

Was this answer helpful?

Yes
No
Thank you for your response

What selection mechanism does the PichiaPink™ Yeast Expression System use?

Product FAQ

Answer

The PichiaPink™ system relies on selection of transformants using ADE2 complementation (i.e., by complementation of adenine auxotrophy) rather than antibiotic selection. The ADE2 gene encodes phosphoribosylaminoimidazole carboxylase, which catalyzes the sixth step in the de novo biosynthesis of purine nucleotides. Mutations in ADE2 lead to the accumulation of purine precursors in the vacuole, which causes the colony to be red in color. In addition, ade2 mutants are adenine auxotrophs that are unable to grow on medium lacking adenine and have a slow growth phenotype on rich medium.

The strains in the PichiaPink™ system are ade2 auxotrophs due to the full deletion of the ADE2 gene and part of its promoter. The PichiaPink™ expression vectors contain the ADE2 gene (under its own promoter) as the selection marker, with the high-copy vectors (pPink-HC and pPinkalpha-HC) containing a truncated ADE2 promoter compared to the full-length ADE2 promoter in the low-copy vector (pPink-LC). Transformation of the PichiaPink™ strains with the expression plasmids enable the strain to grow on medium lacking adenine (Ade dropout medium or minimal medium). Regardless of the host PichiaPink™ strain, both white and slightly pink colonies are obtained on the selection plates upon transformation with the high-copy PichiaPink™ vectors. The color of the colonies indirectly indicates the relative expression levels of the protein of interest as the color of the colony depends on the copy number of the plasmid, which in turn is determined by the promoter strengths of the markers. The pink colonies express very little ADE2 gene product, while the white colonies express higher amounts of the ADE2 gene product, suggesting that those colonies have more copies of the integrated construct. Strains transformed with the low-copy plasmid, pPink-LC, grow faster on medium lacking adenine, generating white colonies due to the stronger promoter on this vector. Since the promoter is stronger, less ADE2 expression is required to allow the strains to grow on medium lacking adenine. As a result, fewer copies of the ADE2 gene/expression construct are required in the strain.

Answer Id: E9482

Was this answer helpful?

Yes
No
Thank you for your response

I am using the pPink-HC vector and the transformation efficiency of the host strain seems to be very low. Why is this?

Product FAQ

Answer

When using the pPink-HC vector, the transformation efficiency of the host strain may appear low because colonies with only a few copies of the marker will not produce enough ADE2 gene product to grow and will be selected against on medium lacking adenine (i.e., adenine dropout medium or minimal medium). Only colonies that have integrated multiple copies of the ADE2 marker will able to grow without adenine. Since the gene of interest is linked to the selection marker, the white colonies could also result in higher expression of the gene of interest.

Answer Id: E9563

Was this answer helpful?

Yes
No
Thank you for your response

My spheroplasting of Pichia worked twice, but hasn't worked since. The OD of the culture simply does not drop.

Product FAQ

Answer

Here are some things to consider:
(1) If the OD of cells that are used is too high, they will not spheroplast. Do not overgrow cells.
(2) Do not use old cells and make sure that they are in log phase of growth.
(3) Make sure to mix zymolase well before using. Zymolase is more of a suspension than a solution.
(4) Make the PEG solution fresh each time and check the pH.

Answer Id: E3737

Was this answer helpful?

Yes
No
Thank you for your response

Will the alpha factor secretion signal work in other yeast?

Product FAQ

Answer

The alpha secretion signal is from S. cerevisiae and is a general yeast secretion signal that has been used in many species including P. pastoris, K. lactis, etc.

Answer Id: E9496

Was this answer helpful?

Yes
No
Thank you for your response