Invitrogen™ PichiaPink™ Media Kit

Catalog No.	Quantity
A11156	1 Kit

Catalog No. A11156 Supplier Invitrogen™ Supplier No. A11156

Description

PichiaPink™ Media Kit

The PichiaPink™ Media Kit is a set of convenient prepackaged media pouches for use with the PichiaPink™ Yeast Expression System. The PichiaPink™ Media Kit contains media needed to grow Pichia pastoris strains from frozen stocks to cultures ready for transformation and selection. The PichiaPink™ Media Kit is an easy way get started using Pichia pastoris for protein production. Use the PichiaPink™ Media Kit to refill your PichiaPink™ Yeast Expression System Kits.

The PichiaPink™ Media Kit comes with prepackaged pouches for the following solutions. Each pouch makes 1 liter of solution. Media pouches should be kept dry and stored at room temperature.

Why Choose the PichiaPink™ Yeast Expression System?
The PichiaPink™ Yeast Expression System is based on the yeast Pichia pastoris. Advantages of Pichia pastoris include rapid growth, well-defined genetic background, simple media formulation, and easy handling. For over 30 years, Pichia pastoris has been used by labs around the world for producing hundreds of different proteins from many species including human (Ref 1, 2). The PichiaPink™ Yeast Expression System allows convenient and cost-effective protein production from small to large scales.

For information on obtaining a commercial-use license for the PichiaPink™Yeast Expression System, please inquire at outlicensing@lifetech.com.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Related Links

Learn more about the PichiaPink™ Yeast Expression System.

Learn more about our other protein expression systems.

References

1. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45-66. [PubMed]
2. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol. 2002 Aug;13(4):329-32. [PubMed]

Specifications

• Content And Storage:

  Contains pouches sufficient to make 4 basic media:

  2 pouches PAD agar, 2 pouches YPDS, 2 pouches YPD, 2 pouches YPD agar, 1 pouch Dextrose

  
  Store at room temperature

• Form: Powder
• Format: Kit
• Media Type: PAD, YP, YPS
• Preparation Method: Autoclave
• Target Organism Class: P. pastoris
• Final Product Type: Liquid Medium, Agar Plates
• For Use With (Equipment): A11150, A11151, A11152, A11153
• Quantity: 1 Kit
• Shipping Condition: Room Temperature
• Strain Designations: PichiaPink™

Frequently Asked Questions (FAQs)

I inoculated 25 mL of MGY with one colony and grew it overnight, but the OD is not 2-6 as mentioned in your manual. Is there something wrong?

This is likely due to low inoculum. We recommend having a starter culture to use, as it is more accurate than using a colony. You can use the doubling time to calculate the starting OD. In YPD, the doubling time is about 1.5 hours. In MGY, the doubling time is going to be about 3 hours.

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?

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.

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

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.

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.

Is there a functional difference between using peptone or tryptone for Pichia media?

Bacto-Tryptone and Bacto-Peptone are two different and specific types of peptones. Bacto-Tryptone is a slightly poorer nitrogen source, and more of the nitrogen is provided by tyrosine and tryptophan. When comparing the two as components of media for Pichia growth, growth curves may differ slightly, but there should be only minor differences between the two. In Pichia media formulations that include Yeast Nitrogen Base as a primary source of nitrogen, such as BMGY and BMMY, there should be very little or no difference.

What are the components and concentrations for 1X YNB (yeast nitrogen base)?

For the pouches sold as Cat. No. Q30007, each pouch contains reagents to prepare 500 mL of 10X YNB for Pichia media or 1,000 mL of 10X YNB for S. cerevisiae media (since Pichia media is 2X what is used for S. cerevisiae). Below is the formulation for 1X YNB:

Ammonium sulfate, 5 g
Biotin, 0.002 mg
Calcium pantothenate, 0.4 mg
Folic acid, 0.002 mg
Inositol, 2 mg
Niacin, 0.4 mg
Para-aminobenzoic acid, 0.2 mg
Pyridoxine HCl, 0.4 mg
Riboflavin, 0.2 mg
Thiamine HCl, 0.4 mg
Boric acid, 0.5 mg
Copper sulfate, 0.4 mg
Potassium iodide, 0.1 mg
Ferric chloride.6H2O, 0.2 mg
Manganese sulfate.H2O, 0.4 mg
Sodium molybdate.2H2O, 0.2 mg
Zinc sulfate.H2O, 0.4 mg
Potassium phosphate monobasic, 1 g
Magnesium sulfate, 0.5 g
Sodium chloride, 0.1 g
Calcium chloride, 0.1 mg

Can YNB medium (with ammonium sulfate, without amino acids) be autoclaved instead of filter-sterilized?

Yes, YNB medium with ammonium sulfate and without amino acids can be autoclaved for sterilization.

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.

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.

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.

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.

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.

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.

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.

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

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.

Do you have any protocols for Pichia fermentation?

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

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

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.

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

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

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

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

Which method do you recommend using for transformation of Pichia?

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.

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

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.

Spheroplast Kit for Yeast (K172001): 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.

What is the mating genotype of your Pichia strains?

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.

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

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.

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

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.

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

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.

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

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.

What do I do if I see a volume loss during a pilot expression of my Pichia culture?

You can supplement with 10% culture volume of a 5% methanol (in water) solution to regenerate the 0.5% methanol concentration each day.

If there are no Zeocin antibiotic-containing YPD plates readily available, would it be possible to spread Zeocin antibiotic on top of YPD plates and still retain efficient selection of yeast?

Zeocin antibiotic can be spread on top of YPD plates for selection of yeast if necessary. There is a report that this works well when done with 10-15 3 mm glass beads. However, it is recommended that some optimization be performed, since top-spreading may dilute the antibiotic's effectiveness.

Will the alpha factor secretion signal work in other yeast?

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.

How is the alpha factor secretion signal sequence processed?

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.

Will a mammalian secretion signal work in Pichia?

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

What is the codon usage for Pichia?

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

How can I convert OD units to approximate Pichia cells/mL (or density)?

An OD600 of 1 is equivalent to 5 x 10e7 Pichia cells/mL. After overnight (O/N) growth from a colony pick, a Pichia culture generally reaches OD 1.3-1.5 (in 2-5 mL).

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

Doubling time is 2-3.5 hrs: Pichia has a doubling time of about 2 hrs of glycerol. 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.

What is the size of the Pichia genomic DNA?

The Pichia genome is similar to that of other yeast, approximately 1.5 x 107 bp (similar to S. cerevisiae) and contains 4 chromosomes (similar to S. pombe). Reference: Ohi H, Okazaki N, Uno S, Miura M, Hiramatsu R (1998) Chromosomal DNA patterns and gene stability of Pichia pastoris. Yeast 14(10):895-903.

We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1.7 Mb to 3.5 Mb, and total genome size was estimated to be 9.5 Mb to 9.8 Mb; however, chromosome-length polymorphisms existed among four strains.

What selection mechanism does the PichiaPink Yeast Expression System use?

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.

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

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

For Research Use Only. Not for use in diagnostic procedures.