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

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. Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center .

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

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. Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center .

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

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. Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center .

Q: Can antibiotics be used during Pichia fermentation?

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. Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center .

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

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. Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center .

Q: Can I use YPD instead of BMGY-type media for Pichia fermentation?

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. Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center .

Q: What is the advantage of mixed feed in Pichia fermentation?

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. Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center .

Q: What can be used as an acid to adjust the pH of Pichia fermentation media? Do I even need to adjust the pH?

You need not add any acid to Pichia fermentation media. A healthy culture always acidifies the medium. If the pH of the culture is increasing, it is a sign of carbon source depletion or ill health of the culture. Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center .

Question 1

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

Accepted Answer

Generally, large colonies represent transformants containing pPIC6/pPIC6α integrants, while small colonies represent transformants containing pPIC6/pPIC6α non-integrants. These non-integrants have transduced the pPIC6/pPIC6α 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 2

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

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 3

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

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 4

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

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 5

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

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 6

Can antibiotics be used during Pichia fermentation?

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 7

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

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 8

Can I use YPD instead of BMGY-type media for Pichia fermentation?

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 9

What is the advantage of mixed feed in Pichia fermentation?

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 10

What can be used as an acid to adjust the pH of Pichia fermentation media? Do I even need to adjust the pH?

Accepted Answer

You need not add any acid to Pichia fermentation media. A healthy culture always acidifies the medium. If the pH of the culture is increasing, it is a sign of carbon source depletion or ill health of the culture.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 11

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

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 12

Do you have any protocols for Pichia fermentation?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 13

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

Accepted 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 14

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

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 15

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

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 16

Which method do you recommend using for transformation of Pichia?

Accepted Answer

We recommend electroporation for transformation of Pichia. Electroporation yields 10e3 to 10e4 transformants per µg of linearized DNA and does not destroy the cell wall of Pichia. If you do not have access to an electroporation device, you may use the Spheroplast Kit for Yeast(Cat. No. K172001), PEG 1000 protocol (page 78 of the manual), LiCl protocol (page 80 of the manual), or the Pichia EasyComp Transformation Kit (Cat. No. K173001). We do not recommend spheroplasting for transformation of Pichia with plasmids containing 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 17

What is the best method for transforming YACs?

Accepted Answer

The best method for transforming YACs into yeast is the spheroplast method.  Yeast are never transformed with YAC vectors by electroporation--the vectors are too big and get sheared on the way into the cell.

Spheroplast Kit for Yeast - FAQs

When selecting for blasticidin-resistant transformants in the X-33 strain using pPIC6/pPIC6α vectors, why do I get large and small colonies on YPD plates containing 300 µg/ml blasticidin?