Can I use YPD 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, and the richer medium makes it difficult to purify secreted proteins from the medium. The BMGY formulation remedies both of these problems.

Answer Id: E9541

Was this answer helpful?

Yes
No
Thank you for your response

Do I 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 medium. It would serve no purpose, other than maybe help dissolve the salts.

Answer Id: E9542

Was this answer helpful?

Yes
No
Thank you for your response

Can antibiotics be used during Pichia fermentation?

Product FAQ

Answer

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.

Answer Id: E9543

Was this answer helpful?

Yes
No
Thank you for your response

Can the methanol and ammonium hydroxide solutions used to prepare Pichia fermentation medium be autoclaved?

Product FAQ

Answer

No, you cannot autoclave methanol. There are two approaches to this, depending a bit on the size of the bioreactor and the volumes involved. You can either dilute to working concentration and filter-sterilize with a filter suitable for alcohols, or you can just assume that methanol is sterile (it should be) and dilute into sterile water. For the ammonium hydroxide solution, you should also not autoclave it. You can assume the 30% stock solution is sterile (nothing should live in this solution) and dilute into sterile water to the working concentration.

Answer Id: E9544

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

Is there a recommended protocol for fermentation using constitutive expression vectors such as pGAPZ?

Product FAQ

Answer

Use the following high cell density protocol for pGAP clones. Feed carbon until the desired density is reached (300 to 400 g/L wet cell weight (WCW)). If the protein is well-behaved in the fermenter, increase to 300-400 g/L WCW as with methanol inducible clones. These densities can be reached in less than 48 hours of fermentation. We have fermented constitutive expressers on glycerol using these protocols with good results. Some modifications to the Fermentation Basal Salts Medium that you might want to make are:

1) Substitute 2% dextrose for the 4% glycerol in the batch medium.
2) Substitute 40% dextrose for the 50% glycerol in the fed-batch medium.
3) Feed the 40% dextrose at 12 mL/L/hr (Jim Cregg has published data on expression using several carbon sources as substrates; dextrose gave the highest levels of expression).
4) Yeast extract and peptone may be added to the medium for protein stability.

One warning: If you are working with His- strains, they remain His- after transformation with pGAPZ. Fermentation in minimal medium will require addition of histidine to the fermenter.

Answer Id: E9545

Was this answer helpful?

Yes
No
Thank you for your response

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

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

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

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

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

Does Pichia pastoris secrete proteins that can be toxic to itself or to other cells?

Product FAQ

Answer

Certain yeast strains secrete a protein toxin, which inhibits the growth of sensitive pathogens and yeasts. Studies have shown that production of the toxin is dependent on the presence of linear, double-stranded DNA plasmids in the killer yeasts. In the yeast Pichia pastoris, two linear double-stranded DNA plasmids have been identified. In the publication listed below, the search for toxin-producing capability in P. pastoris was conducted and no killer activity could be detected when 14 different indicator strains were tested. Reference: Banerjee and Verma (2000) Search for a Novel Killer Toxin in Yeast Pichia pastoris. Plasmid 43:181-183.

Answer Id: E9511

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

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

What pattern of oxygen uptake should I expect to observe during a Pichia fermentation run?

Product FAQ

Answer

It depends whether the clone is Mut+ or a MutS.

For a Mut+ clone, you should expect that initially (in the first 2-4 hours of induction), the oxygen uptake rate of the culture would be lower than that at the end of the glycerol batch phase. After the culture becomes adapted to methanol, the oxygen uptake rate will significantly increase, if the culture is healthy (i.e., not poisoned by too much methanol). One should run methanol spike tests during fermentation of Mut+ clones.

For a MutS clone, one can expect that the oxygen uptake rate will be lower than that at the end of the glycerol batch phase throughout most of the fermentation. One has to be very careful not to poison MutS clones.

Answer Id: E9538

Was this answer helpful?

Yes
No
Thank you for your response