You searched for: 

A11153

Product FAQ

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

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

Product FAQ

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

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

Product FAQ

What is the mating genotype of your Pichia strains?

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

Product FAQ

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

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

Product FAQ

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

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

Product FAQ

Do I need to add sulfuric acid to the fermentation PTM trace salts?

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

Product FAQ

Will the alpha factor secretion signal work in other yeast?

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

Product FAQ

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

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

Product FAQ

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

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

Product FAQ

What is the advantage of mixed feed in Pichia fermentation?

Answer

The use of mixed feeds is mainly due for "turning down" the level of expression for proteins that are troublesome for Pichia. We have generally used mixed feeds for MutS clones. The idea is to keep the culture in a state of more active growth, and thus "happier" to express proteins.

Answer Id: E9540

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Will a mammalian secretion signal work in Pichia?

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

Product FAQ

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

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

Product FAQ

Can antibiotics be used during Pichia fermentation?

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

Product FAQ

Will Pichia pastoris vectors (e.g., pPICZ, pPIC6, pPIC9K, pPIC3.5K, pAO815) work in Pichia methanolica? Is the TEF1 promoter functional in Pichia methanolica?

Answer

No, Pichia pastoris vectors will not work in Pichia methanolica; both Pichia pastoris and Pichia methanolica vectors have promoters derived from alcohol oxidase but they are not homologous, so the Pichia pastoris vectors will not be able to integrate or replicate in Pichia methanolica. The TEF1 promoter is probably functional in Pichia methanolica.

Answer Id: E9509

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

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