Will a recombinant protein be glycosylated as it goes through the secretory pathway in a yeast cell?

Product FAQ

Answer

A secreted protein will be exposed to the glycosylation machinery and might be glycosylated if the protein contains the standard N-linked or O-linked glycosylation amino acid consensus sequence.

Answer Id: E9502

Was this answer helpful?

Yes
No
Thank you for your response

Is it critical that one uses PEG 4000 for yeast transformations?

Product FAQ

Answer

PEG 4000 seems to work best for yeast transformations, although PEG 3350 has been used in-house with success.

Answer Id: E9530

Was this answer helpful?

Yes
No
Thank you for your response

When doing Pichia expression from a plasmid containing the Zeocin™ antibiotic resistance gene, is it necessary to have Zeocin™ antibiotic in the expression medium?

Product FAQ

Answer

There is no need for maintaining Zeocin™ antibiotic selection in the Pichia expression medium, since Pichia pastoris transformants are stable integrants with the gene of interest integrated into the genome.

Answer Id: E9503

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

Is there an electroporation protocol for Pichia cells that doesn't require starting with 500 mL of cells?

Product FAQ

Answer

The following protocol has been used numerous times for Pichia pastoris. It uses a 250 mL culture that is eventually scaled down to 1 mL aliquots of each strain.

- Inoculate 10 mL YPD media with Pichia strain and grow O/N, shaking at 30 degrees C.
- In the morning, check the OD600. To get them in log phase by the afternoon, dilute cells to hit an OD600 of approximately 3.0 at 4 or 5 pm.
- When the OD600 reaches approximately 3.0, inoculate 250 mL of YPD with 250 μL of culture. The objective is to have healthy, log-phase cells in the morning at an OD600 of around 1.0.
- If the OD600 is ~1.0, spin the cells in a 1 L bottle at 3K rpm for 10 minutes.
- Gently resuspend in 250 mL cold dH20.
- Transfer to a 500 mL centrifuge bottle and spin at 3K for 10 min. Repeat.
- Resuspend in 20 mL cold 1 M sorbitol and transfer to a 50 mL conical tube.
- Spin at 3K rpm for 10 min.
- Resuspend in 1 mL 1M sorbitol, and keep on ice.
- Use 80 μL of host strain for each electroporation.

Answer Id: E9532

Was this answer helpful?

Yes
No
Thank you for your response

What is the mating genotype of your Pichia strains?

Product FAQ

Answer

All of our Pichia strains are homothallic strains. This means that they actually switch mating type with each generation. In Saccharomyces strains, this would lead to the culture rapidly becoming entirely diploid. In contrast, Pichia pastoris strains mate inefficiently to form diploids. Therefore, at any given time, the cells in the population are both “a” and “alpha” mating types.

Answer Id: E9522

Was this answer helpful?

Yes
No
Thank you for your response

Do you have any protocols for Pichia fermentation?

Product FAQ

Answer

We do not offer any protocols for Pichia fermentation. Please refer to the document titled “Pichia Fermentation Guidelines” on our website.

Answer Id: E9533

Was this answer helpful?

Yes
No
Thank you for your response

Can the ProBond™ Purification system be used with Pichia lysates?

Product FAQ

Answer

Yes, you can use ProBond™ with His-tagged proteins expressed in Pichia. Here are some suggestions for using ProBond with Pichia supernatant:
1. Adjust pH of Pichia supernatant to 7.5-8.0.
2. Decant the supernatant from the heavy white precipitate. It is recommended to keep the precipitate for later solubilization in the rare case where the expressed protein has co-precipitated.
3. Centrifuge the supernatant to remove leftover cell debris or other material that might clog the column.
4. Adjust the conductivity to that of 500 mM NaCl with salt addition (may not be required since Pichia media is high salt).
5. Run the column according to the instructions in the manual.

Answer Id: E4326

Was this answer helpful?

Yes
No
Thank you for your response

What is the Nitrogen source in the media for fermentation?

Product FAQ

Answer

During the Pichia fermentation process, the nitrogen source is the ammonium hydroxide used to adjust the pH. There is no nitrogen in the basal or trace salts.

Answer Id: E3751

Was this answer helpful?

Yes
No
Thank you for your response

What is the doubling time of Pichia? How long should I wait to see colonies on agar?

Product FAQ

Answer

Pichia has a doubling time of about 2-3.5 hours in SC media with glucose. The yeast grow slowly at 30 degrees C and it takes at least 3 days for colonies. In practice, it takes anywhere from 3 to 7 days to get nice-sized colonies.

Answer Id: E9489

Was this answer helpful?

Yes
No
Thank you for your response

Are Invitrogen™ Pichia strains haploid or diploid? Can they sporulate?

Product FAQ

Answer

Pichia pastoris most commonly exists in a vegetative haploid state. On nitrogen limitation, mating can occur and diploid cells are formed. Since cells of the same strain can readily mate with each other, P. pastoris is by definition homothallic. Relative to Saccharomyces cerevisiae, which is heterothallic, the haploid state of P. pastoris is more stable. Under nitrogen limiting conditions P. pastoris diploids proceed through meiosis to the production of asci containing four haploid spores.

Answer Id: E3916

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:

- If the OD of cells that are used is too high, they will not spheroplast. Do not overgrow cells.
- Do not use old cells and make sure that they are in log phase of growth.
- Make sure to mix zymolyase well before using. Zymolyase is more of a suspension than a solution.
- Make the PEG solution fresh each time and check the pH.

Answer Id: E9560

Was this answer helpful?

Yes
No
Thank you for your response

What are the different methods available for transformation of Pichia, and how do they compare?

Product FAQ

Answer

Here are the different methods available for Pichia transformation:

Pichia EasyComp™ Transformation Kit: easy-to-use, ready-made reagents
This method produces chemically competent Pichia cells and provides a rapid and convenient alternative to electroporation. Transformation efficiency is low (transformation of 50 μl of competent cells with 3 μg of linearized plasmid DNA yields about 50 colonies), and hence it is very difficult to isolate multi-copy integrants. Higher transformation efficiencies are often obtained with frozen versus freshly prepared cells.

PEG 1000 transformation: easy, do-it-yourself protocol
It is critical to add DNA to frozen cell samples, as cell competence decreases very rapidly after the cells thaw-even when held on ice. To perform multiple transformations, it is recommended to process them in groups of six at a time. The PEG method is usually better than LiCl, but not as good as spheroplasting or electroporation for transformation. However, it is convenient for people who do not have an electroporation device. The transformation efficiency is 10e2 to 10e3 transformants per mg of DNA.

Lithium chloride transformation: easy, do-it-yourself protocol
This method is an alternative to transformation by electroporation. Competent cells must be made fresh. Transformation efficiency is 10e2 to 10e3 transformants per μg linearized DNA. Note: Lithium acetate does not work with Pichia pastoris. Use only lithium chloride.

Electroporation: easy and high efficiency, do-it-yourself protocol; does not destroy the cell wall
Competent cells must be made fresh. Transformation efficiency is 10e3 to 10e4 transformants per ™μg of linearized DNA.

Pichia Spheroplast Kit: cell wall digested to allow DNA to enter the cell; the procedure involves treating cells with zymolyase to create spheroplasts.
You must determine the optimal time to treat with zymolyase by taking OD600 readings at increasing time points. Longer incubations with zymolyase result in reduced transformation efficiency. Spheroplasts are combined with DNA and then plated. Transformation efficiency is 10e3 to 10e4 transformants per μg of linearized DNA. Note: Spheroplasting is not recommended for Pichia vectors with an antibiotic resistance marker. Damage to the cell wall leads to increased sensitivity to the antibiotic, causing putative transformants to die before they express the antibiotic resistance gene. In contrast, spheroplasting can be used for transformation of PichiaPink™ vectors, because these vectors are selected using auxotrophic markers.

Answer Id: E9525

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

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

Product FAQ

Answer

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

For a Mut+ you should expect that initially (in the first 2-4 hours of induction) the oxygen uptake rate of the culture would be lower than the end of 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 one can expect that the oxygen uptake rate will be lower than the end of glycerol batch phase through out most of the fermentation. One has to be very careful not to poison MutS clones.

Answer Id: E3752

Was this answer helpful?

Yes
No
Thank you for your response