Are Invitrogen™ Pichia strains haploid or diploid? Can they sporulate?
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: 3916
How can I freeze Pichia?
Store glycerol stocks frozen at -80° C in 15% glycerol. Glycerol stocks are good indefinitely (unless there are numerous freeze-thaws). When making a glycerol stock, we recommend using an overnight culture and concentrating it 2-4 fold. Spin down cells and suspend in 25-50% of the original volume with glycerol/medium. It is better to store frozen cells in fresh medium plus glycerol, rather than simply adding glycerol into the overnight culture.
Answer Id: 4227
Can I express complex proteins or one with a quaternary structure in Pichia pastoris?
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: 4244
Can YNB media (with Ammonium Sulfate without Amino Acids) be autoclaved instead of filter sterilized?
Can the methanol and ammonium hydroxide solutions used to prepare Pichia fermentation media 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.
Answer Id: 4271
Does Invitrogen™ have protocols or recommendations for Selenomethionine incorporation by Pichia to allow structural studies of crystallized protein?
Jim Cregg (Keck Graduate Institute, developer of Pichia expression system for Phillips Petroleum), Senyon Choe (head of Structural Biology at The Salk Institute) and Life Technologies scientists have been unable to achieve adequate protein yields with greater than 40-50% incorporation of Selenomethionine (Se-Met), and unfortunately this level is not useful for phasing of crystals for x-ray analysis. The fundamental problem for Se-Met incorporation is that Met auxotrophic strains of Pichia, unlike Met auxotrophic strains of E. coli, do not grow on Se-Met. A number of groups have even attempted second site suppression mutagenesis in order to find genetic backgrounds that are Met auxotrophic but can grow (although poorly) on Se-Met. This has not been successful. Currently, Met WT strains of Pichia incorporate too much unmodified Met into proteins to produce protein that is >90% Se-Met substituted, which appears to be necessary for adequate phasing.
Some promising references regarding this issue are:
1. Larsson, et al. Preparation and crystallization of selenomethionyl dextranase from Penicillium minioluteum expressed in Pichia pastoris. Acta Crystallogr D Biol Crystallogr. 2002 Feb;58(Pt 2):346-8.
2. Larsson, et al. Dextranase from Penicillium minioluteum: reaction course, crystal structure, and product complex. Structure (Camb). 2003 Sep;11(9):1111-21.
3. Xu, et al. Crystallization and X-ray analysis of native and selenomethionyl beta-mannanase Man5A from blue mussel, Mytilus edulis, expressed in Pichia pastoris. Acta Crystallogr D Biol Crystallogr. 2002 Mar;58(Pt 3):542-5.
Answer Id: 4284
Can the ProBond™ Purification system be used with Pichia lysates?
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: 4326
Can old premixed lithium acetate buffers be used for preparing and transforming Saccharomyces cerevisiae?
Stock buffers of TE, Lithium Acetate, and PEG can be stored. However the combined solution which is used to prepare the cells for transformation must be made fresh every time. There is a loss in transformation efficiency if the solutions are not freshly prepared.
Answer Id: 3732
Will a mammalian secretion signal work in Pichia?
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:
(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: 3737
Will a recombinant protein be glycosylated as it goes through the secretory pathway in a yeast cell?
If a protein goes through the secretory pathway--a path that the alpha factor leader sequence or any other secretion signal directs such proteins to take--it will be exposed to the glycosylation machinery and it might be glycosylated. However, the standard N-linked or O-linked glycosylation amino acid consensus sequences must be present in the expressed protein.
Answer Id: 3738
What causes the media to turn green during Pichia fermentation?
The green color found in the supernatant of methanol grown, high cell density Pichia fermentations is due to alcohol oxidase (Aox1p). Aox1p assembles into a homo-octamer and binds flavin adenine dinucleotide as a prosthetic group in vivo. At high concentrations, the enzyme can form a crystalloid to produce a chromophor that is green. Up to 30% of total cell protein is Aox1p, when Pichia cells are grown on methanol. Aox1p, while not a secreted enzyme, will accumulate in the culture supernatant due to leakage from the cells or lysis.
Pichia can be grown to the same high densities on other carbon sources such as glycerol or dextrose without developing this color. The alcohol oxidase production is either uninduced or tightly repressed under these conditions.
To successfully purify a protein away from Aox1p (and FAD), you can use either a S column (reverse cation exchange column), or a Q column (Sepharose Fast Flow ion exchange column). The Q column is not always as efficient as an S column unless it is a large diameter and long column. Purification of His-tagged proteins on ProBond would also effectively remove the Aox1p.
Answer Id: 3739
How can I convert OD units to approximate Pichia cells/ml (or density)?
Does Pichia secrete proteases in the media? What protease inhibitors can be used to prevent proteolysis?
Yeasts in general are known to secrete proteases. There are some proteins specifically susceptible to proteases that have optimal activity at neutral pH. If this is the case, expression using unbuffered media may be indicated. As Pichia expression progresses in an unbuffered medium such as MMH (minimal methanol plus histidine), the pH drops to 3 or below, inactivating many neutral pH proteases. Although the acidic environment of the culture should prevent activity of neutral proteases, you may use PMSF and EDTA at a 1mM concentration in Pichia crude supernatant (refresh the PMSF every few hours) and then monitor for protease activity. See Deutscher(1990) Guide to Protein Purification, Methods in Enzymology for details.
In contrast, it has been reported that by including 1% Casamino acids (Difco) and buffering the medium at pH 6.0, extracellular proteases were inhibited, increasing the yield of mouse epidermal growth factor. Please see Clare, J. J. et al. (1991) Gene 105:205-212.
Additionally, major vacuolar proteases may be a factor in degradation, particularly in fermentor cultures that have the combination of the high cell density and lysis of a small percentage of cells. Using a host strain that is defective in these proteases may help reduce degradation. SMD1168 and SMD1168H are protease-deficient Pichia strains that are defective for Pep4p, a proteinase that is required for the activation of other vacuolar proteases, such as carboxypeptidase Y and proteinase B. Please see Higgins D. R. and Cregg, J. M. (1998) Pichia Protocols. Humana Press, Totowa, New Jersey. Please note that SMD1168, SMD1168H and the Pichia Protocols book can be ordered from Invitrogen™.
Answer Id: 3749
What anti-foam can you use with Pichia fermentation?
Most researchers prefer MAZU DF 204 or KFO 673. Antifoam 289 may be used, though it will not last very long. Use the minimum amount of antifoam to control foaming. A healthy five liter fermentation will require from 0 to a few milliliters of antifoam. Preferably add via a well regulated antifoam controller. If the culture foams excessively it is a sign of carbon source limitation, low pH, or ill health of the culture.
Answer Id: 3750