You searched for: 

A11151

Product FAQ

How is the alpha factor secretion signal sequence processed?

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

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

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

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?

Answer

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.

Answer Id: E9497

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

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

Product FAQ

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

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

Product FAQ

What is the size of the Pichia genomic DNA?

Answer

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.

Answer Id: E9488

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

Answer

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

Answer Id: E9499

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the codon usage for Pichia?

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

Product FAQ

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

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

Product FAQ

What choices do you offer for protein expression in a yeast host system, and what are their features?

Answer

We offer the original Pichia pastoris expression systems, PichiaPink™ expression system, and Saccharomyces cerevisiae yeast expression system for expression of recombinant proteins. Both P. pastoris and S. cerevisiae have been genetically well-characterized and are known to perform many posttranslational modifications.

The P. pastoris expression system combines the benefits of expression in E. coli (high-level expression, easy scale-up, and inexpensive growth) and the advantages of expression in a eukaryotic system (protein processing, folding, and posttranslational modifications), thus allowing high-level production of functionally active recombinant protein. As a yeast, Pichia pastoris shares the advantages of molecular and genetic manipulations with Saccharomyces cerevisiae, and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia pastoris very useful as a protein expression system. The Pichia expression vectors contain either the powerful alcohol oxidase (AOX1) promoter for high-level, tightly controlled expression, or the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for high-level, constitutive expression. Both inducible and constitutive expression constructs integrate into the P. pastoris genome, creating a stable host that generates extremely high protein expression levels, particularly when used in a fermentor. The Pichia pastoris expression systems we offer include:

- PichiaPink™ Yeast Expression System: Newer Pichia pastoris expression system that contains both low- and high-copy plasmid backbones, 8 secretion signal sequences, and 4 yeast strains to help optimize for the highest yield possible of the recombinant protein. All PichiaPink™ vectors contain the AOX1 promoter for high-level, inducible expression and the ADE2 marker for selecting transformants using ADE2 complementation (i.e., by complementation of adenine auxotrophy) rather than antibiotic selection. However, they express the ADE2 gene product from promoters of different lengths, which dictate the copy number of the integrated plasmids. The pPink-LC vector has an 82 bp promoter for the ADE2marker and offers low-copy expression, and the pPink-HC vector has a 13 bp promoter for the ADE2marker and offers high-copy expression. The system also includes the pPinkalpha-HC vector (containing S. cerevisiae alpha-mating factor pre-sequence) for high copy number secreted expression, and provides eight secretion signal sequences for optimization of secreted expression.
- EasySelect™ Pichia Expression Kit: One of the original Pichia expression kits that contains the pPICZ and pPICZalpha vectors, for intracellular and secreted expression, respectively, of the gene of interest. These vectors contain the AOX1 promoter for high-level, inducible expression and the Zeocin™ antibiotic resistance marker for direct selection of multi-copy integrants. They facilitate simple subcloning, simple purification, and rapid detection of expressed proteins.
- Original Pichia Expression Kit: The kit includes the pPIC9, pPIC3.5, pHIL-D2, and pHIL-S1 vectors, each of which carries the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. pPIC9 carries the S. cerevisiae alpha-factor secretion signal while pHIL-S1 carries the Pichia pastoris alkaline phosphatase signal sequence (PHO) to direct transport of the protein to the medium. pHIL-D2 and pPIC3.5 are designed for intracellular expression.
- Multi-Copy Pichia Expression Kit: This kit is designed to maximize expression and contains the pPIC3.5K, pPIC9K, and pAO815 vectors, which allow production and selection of Pichia strains that contain more than one copy of the gene of interest. They allow isolation and generation of multicopy inserts by in vivo methods (pPIC3.5K and pPIC9K) or in vitro methods (pAO815). All of these vectors contain the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K and pAO815 remain intracellular. The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance marker that confers resistance to Geneticin™ Reagent in Pichia. Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin™ Reagent. Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to Geneticin™ Reagent. The ability to grow in high concentrations of Geneticin™ indicates that multiple copies of the kanamycin resistance gene and the gene of interest are integrated into the genome.
- For expression in S. cerevisiae, we offer the pYES™ Vector Collection. Each pYES™ vector carries the promoter and enhancer sequences from the GAL1 gene for inducible expression. The GAL1 promoter is one of the most widely used yeast promoters because of its strong transcriptional activity upon induction with galactose. pYES™ vectors also carry the 2m origin and are episomally maintained in high copy numbers (10-40 copies per cell).

Answer Id: E9478

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

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

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

 Can I express complex proteins or one with a quaternary structure in Pichia pastoris? 

Answer

Pichia is capable of correctly assembling proteins with a quaternary structure. One of the earliest proteins to be expressed in Pichia was the Hepatitis B Surface antigen which was assembled in its natural form, the 22nm particle (reference: Cregg (1987). "High-level expression and efficient assembly of Hepatitis B surface antigen in the methylotrophic yeast P. Pastoris". Biotechnology 5:479-485.)  In consideration of the particle assembly problem, Cregg postulated that one or more post-translational events important in the formation of particles may be slow relative to the synthesis of HBsAg protein. Therefore, he used mutS since it has a slower growth rate.

Answer Id: E4244

Was this answer helpful?

Yes
No
Thank you for your response