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General

Yeast is a single-celled, eukaryotic organism that can grow quickly in defined media (doubling times are typically 2.5 hr in glucose-containing media) and is easier and less expensive to use for recombinant protein production than insect or mammalian cells (see table below). These positive attributes make yeast suitable for use in formats ranging from multi-well plates, shake flasks, and continuously stirred tank bioreactors to pilot plant and industrial-scale reactors.

The most commonly employed species in the laboratory are Saccharomyces cerevisiae (also known as Baker’s or Brewer’s yeast) and some methylotrophic yeasts of the Pichia genus. Both S. cerevisiae and P. pastoris have been genetically characterized and shown to perform the posttranslational disulphide bond formation and glycosylation that is crucial for the proper functioning of some recombinant proteins. However, it is important to note that yeast glycosylation does differ from that in mammalian cells: in S. cerevisiae, O-linked oligosaccharides contain only mannose moieties, whereas higher eukaryotic proteins have sialylated O-linked chains. Furthermore S. cerevisiae is known to hyperglycosylate N-linked sites, which can result in altered protein binding, activity, and potentially yield an altered immunogenic response in therapeutic applications. In P. pastoris, oligosaccharides are of much shorter chain length and a strain has been reported that can produce complex, terminally sialylated or “humanized” glycoproteins.

Characteristics

E. coli

Yeast

Insect Cells

Mammalian Cells

Cell growth

Rapid (30 min)

Rapid (90 min)

Slow (18–24 hr)

Slow (24 hr)

Complexity of growth medium

Minimum

Minimum

Complex

Complex

Cost of growth medium

Low

Low

High

High

Ease of use

Easy

Easy to medium Complex

Complex

Complex

Expression level

High

Low–high

Low–high

Low–moderate

Extracellular expression

Secretion to periplasm

Secretion to medium

Secretion to medium

Secretion to medium

Posttranslational modifications

E. coli

Yeast

Insect Cells

Mammalian Cells

Protein folding

Refolding usually required

Refolding may be required

Proper folding

Proper folding

N-linked glycosylation

None

High mannose

Simple, no sialic acid

Complex

O-linked glycosylation

No

Yes

Yes

Yes

Phosphorylation

No

Yes

Yes

Yes

Acetylation

No

Yes

Yes

Yes

Acylation

No

Yes

Yes

Yes

Gamma-carboxylation

No

No

No

Yes

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 pPinkα-HC vector (containing S. cerevisiae α-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 pPICZα 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 α-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).

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. 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. In S. cerevisiae, replicating plasmids are used for episomal expression whereas in Pichia pastoris, plasmids are integrated into the host chromosome.

In comparison to Saccharomyces cerevisiae, Pichia may have an advantage in the glycosylation of secreted proteins because it may not hyperglycosylate. Both Saccharomyces cerevisiae and Pichia pastoris have a majority of N-linked glycosylation of the high-mannose type; however, the length of the oligosaccharide chains added posttranslationally to proteins in Pichia (average 8–14 mannose residues per side chain) is much shorter than those in Saccharomyces cerevisiae (50–150 mannose residues). Very little O-linked glycosylation has been observed in Pichia. In addition, Saccharomyces cerevisiae core oligosaccharides have terminal α1,3 glycan linkages whereas Pichia pastoris does not. It is believed that the α1,3 glycan linkages in glycosylated proteins produced from Saccharomyces cerevisiae are primarily responsible for the hyper-antigenic nature of these proteins, making them particularly unsuitable for therapeutic use. Although not yet proven, this is predicted to be less of a problem for glycoproteins generated in Pichia pastoris, because it may resemble the glycoprotein structure of higher eukaryotes.

We recommend storing yeast 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.

Pichia Expression Systems

PichiaPink™ Yeast Expression System offers significant advantages compared to the original EasySelect™ Pichia system, and they are listed below:

 

PichiaPink™ Yeast Expression System

EasySelect™ Yeast Expression System

Plasmid copy number

Both high and low copy enables optimization of toxic protein expression

High copy number only

No. of secretion signal leader sequences

8

1

No. of strains

4

3

No. of protease-deficient host strains

3

0

Antibiotic resistance marker

None, relies on adenine selection

Yes, uses Zeocin™ antibiotic

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

pPinkα-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. 

The EasySelect™ Pichia Expression Kit features multicopy selection with Zeocin™ antibiotic selection and can do almost everything the Multi-Copy Pichia Expression Kit can do. The pAO815 vector of the Multi-Copy kit is uniquely suited for in vitro multimerization. The other Multi-Copy vectors have the kanamycin resistance gene that expresses at low level (confers resistance to Geneticin® in yeast).

The EasySelect™ Pichia Expression Kit allows easy and improved selection with Zeocin™ antibiotic instead of the HIS marker. The kit also comes with the EasyComp™ Pichia Transformation Kit instead of the Pichia Spheroplast Kit.

Kex2 cleaves the alpha secretion signal peptide from the N-terminus of the overexpressed protein in yeast. While Ste13 generally removes the Glu-Ala amino acids, which are between the Kex2 site and your experimental protein, the Ste13 cleavage may not be complete. To ensure that the N-terminus of your protein is exposed after Kex2 cleavage, you may clone your gene of interest flush with the Kex2 cleavage site. In order to clone your gene flush with the Kex2 cleavage site, you must cut the pPICα vector with XhoI (and any other appropriate enzyme for cloning in-frame with the 3' end of the vector). By cutting the vector with XhoI alone, you will excise 62–67 nucleotides of the vector (depending on which vector you are using), including the Kex2 signal cleavage site. In order to regenerate this signal cleavage site, you will need to generate a forward primer that contains the XhoI site, followed by the Kex2 cleavage sequence and nucleotides from your gene of interest.

After amplification of your gene, you will digest your PCR product with XhoI and, if necessary, with a second restriction enzyme, which will keep your gene of interest in-frame with the C-terminal tag.

Please note: The Glu-Ala-Glu-Ala sequence may not be completely essential for cleavage, but it does have an influence. In general, the efficiency of cleavage by the various kexins is affected by sequences both upstream and downstream of the Lys-Arg dibasic motif, but it seems to vary from one member to another. N-terminal sequences may not matter, but amino acid sequences that confer secondary structure that obscures the cleavage site may cause a problem. A number of amino acids are tolerated after the KEX2 cleavage site: these include aromatic amino acids, small amino acids, histidine, and methionine. Proline will inhibit the activity of KEX2.

The Pichia methanolica Expression System has been discontinued.

Yes, we do offer the pFLD and pFLDα vectors for high-level, inducible expression in Pichia with methanol, methylamine, or methanol plus methylamine.

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.

Pichia has a doubling time of about 2–3.5 hours in SC media with glucose. The yeast grow slowly at 30°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.

An OD600 of 1 is equivalent to 5 x 107 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).

The kanamycin resistance gene was cloned into pPIC9K with its native promoter. It was not cloned with a Pichia or other yeast promoter, meaning the level of expression is extremely low. Therefore, multicopy integrants are required to obtain resistance to G418.

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

pTEF1/Zeo is a ZeoCassette™ vector that contains the Zeocin™ antibiotic resistance gene under control of the S. cerevisiae TEF1 promoter and the bacterial EM7 promoter. The ZeoCassette™ is flanked by polylinkers that can be used to excise the ZeoCassette™ for use in a different vector. Alternatively, different elements can be cloned into the ZeoCassette™ vector backbone to develop a new vector. The Saccharomyces TEF1 promoter is active in Pichia pastoris.

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

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.

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.

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.

Expression levels are entirely protein-dependent. Please refer to page 60 of the EasySelect™ manual that lists literature reports for various proteins. The list includes expression levels and type of strain used. The list ranges from 0.001 to 12 grams per liter. Higher expression levels can be achieved with fermentation.

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

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 22 nm particle. (Reference: Cregg JM et al. (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast P. Pastoris. Nat Biotechnol 5:479–485.) In consideration of the particle assembly problem, Cregg postulated that one or more posttranslational 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.

The major advantage of expressing recombinant proteins as secreted proteins is that Pichia pastoris secretes very low levels of native proteins. Since there is a very low amount of protein in the minimal Pichia growth medium, this means that the secreted heterologous protein comprises the vast majority of the total protein in the medium and serves as the first step in purification of the protein.

Note: 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.

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.

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.

We recommend the following guidelines for optimal pPICZ-lacZ expression:

1) Grow a 20 mL culture to OD600 of 8.0 in BMGY.
2) Concentrate into 5 mL BMMY. Induce with methanol.
3) Take time points every day.
4) Harvest cells and lyse into 500 μL breaking buffer or SDS-PAGE sample buffer.

Add 5% ammonium hydroxide solution to maintain the pH of a Pichia culture grown in shake flasks. It is used at a 28% concentrated form in fermentors. Ammonium hydroxide also acts as a nitrogen source for Pichia cells.

Under the conditions in which Pichia is fermented (aerobically), ethanol is not produced. Oxidative metabolism of methanol first produces formaldehyde, which is then converted to carbon dioxide. There is an assimilatory cycle involving formaldehyde too, but no ethanol is made in this pathway, either.

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 1 mM 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 JJ 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 DR and Cregg JM (1998) Pichia Protocols, Humana Press, Totowa, New Jersey. Please note that SMD1168, SMD1168H, and the Pichia Protocols book can be ordered from us.

In the following reference, 1% casamino acids were used: Clare JJ et al. (1991) Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105(2):205–212.

In this paper, the researchers found that although Pichia grew to a similar cell density in both YP and YNB, only a very low level of mouse epidermal growth factor (0.07 µg/mL) was present in supernatants from single-copy transformants when grown in YNB, and this decreased during further incubation. By using YNB medium that had been buffered to pH 6.0 and supplemented with 1% casamino acids, secreted mEGF levels substantially increased to ~1.9 µg/mL for single-copy transformants.

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.

The molecular weight of the AOX1 gene product is 72 kDa (Reference: Ellis SB, Brust PF, Koutz  PJ, Waters  AF, Harpold MM, Gingeras TR (1985) Isolation of Alcohol Oxidase and Two other Methanol Regulatable Genes from the Yeast, Pichia pastoris. Mol. Cell. Biol. 5: 1111-1121). The AOX2 gene product is also 72 kDa.

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 43:181­183. 

The lyticase for our protocol is crude lyticase. A pure preparation is more expensive and it is not necessary. We routinely use zymolyase in this protocol for lysing the cells (5 µL of a 1 mg/mL stock). This is excessive, but it works fine. Reference: BioTechniques 20:980–982, June 1996.

500 units of lyticase must be used to achieve similar effects to 1 unit of zymolyase. To lyse 1 mL of Pichia cells for PCR analysis, 25 total units of lyticase were used compared to 0.05 units of zymolyase. Zymolyase is available in different purities. 20T zymolyase should be sufficient for all purposes. The further purified 100T should not be necessary.

Yes, you can use the ProBond™ system with His-tagged proteins expressed in Pichia. Here are some suggestions for using the ProBond™ system with Pichia supernatant: 

  1. Adjust the pH of the 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.

HSA will run at 55 kD on a non-reducing SDS-PAGE gel and at about 66 kDa on a reducing SDS-PAGE gel.

Pichia Strains and Transformation

Here are the Pichia strains we offer with their genotype and phenotype features:

Pichia pastoris Strains

Protease Wild-Type Strains Genotype Phenotype Application
X-33 Wild-type Mut+ His+ Selection of Zeocin™ antibiotic-resistant expression vectors
GS115 his4 Mut+ His- Selection of expression vectors containing HIS4
KM71H aox1::ARG4, arg4 MutS His+ Selection of Zeocin™ antibiotic-resistant expression vectors to generate strains with MutS phenotype
Protease-Deficient Strains Genotype Phenotype Application
SMD1168 his4, pep4 Mut+ His- Selection of expression vectors containing HIS4 in a strain without proteinase A activity
SMD1168H pep4 Mut+ His+ Selection of Zeocin™ antibiotic-resistant expression vectors in a strain without proteinase A activity

PichiaPink™ Strains

Protease Wild-Type Strain

Genotype

Phenotype

Application

PichiaPink™ Strain 1

ade2

(ura5Δ::ScSUC2 ade2Δ::lacZ-URA5-lacZ)

Mut+ Ade2-

Pink/white selection of PichiaPink™ expression vectors

Protease-Deficient Strains

Genotype

Phenotype

Application

PichiaPink™ Strain 2

ade2, pep4

(ura5Δ::ScSUC2 pep4Δ::lacZ ade2Δ::lacZ-URA5-lacZ)

Mut+ Ade2-

Pink/white selection of PichiaPink™ expression vectors in a strain without proteinase A activity

PichiaPink™ Strain 3

ade2, prb1

(ura5Δ::ScSUC2 prb1Δ::lacZ ade2Δ::lacZ-URA5-lacZ)

Mut+ Ade2-

Pink/white selection of PichiaPink™ expression vectors in a strain without proteinase B activity

PichiaPink™ Strain 4

ade2, prb1, pep4

(ura5Δ::ScSUC2 pep4Δ::lacZ prb1Δ::lacZ ade2Δ::lacZ-URA5-lacZ)

Mut+ Ade2-

Pink/white selection of PichiaPink™ expression vectors in a strain without proteinase A and proteinase B activity

Upon receipt, we recommend storing Pichia strain stabs at 4°C. For long-term storage, we recommend preparing a glycerol stock (in 15% glycerol) immediately upon receipt and storing at –80°C. 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.

The only MutS Pichia strain we offer is KM71H, Cat. No. C18200. We used to offer KM71, but it has been discontinued.

The advantage of using KM71H is that there is no need to screen for the Mut phenotype on methanol minimal medium. All transformants will be MutS. In some cases, MutS strains will lead to higher expression compared to a Mut+ strain. 

Pichia pastoris most commonly exists in a vegetative haploid state. Upon 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.

Two genes in Pichia pastoris code for alcohol oxidase—AOX1 and AOX2. The AOX1 gene product accounts for the majority of alcohol oxidase activity in the cell. Expression of the AOX1 gene is tightly regulated and induced by methanol to very high levels. The AOX1 protein typically accounts for ≥30% of the total soluble protein in cells grown on methanol. While AOX2 is about 97% homologous to AOX1, growth on methanol is much slower than with AOX1. Loss of the AOX1 gene, and thus a loss of most of the cell's alcohol oxidase activity, results in a strain with a MutS (methanol utilization slow) phenotype. A MutS strain has a mutant aox1 locus, but is wild-type for AOX2. It has reduced ability to metabolize methanol and thus exhibits poor growth on methanol medium. MutS has in the past been referred to as Mut–. Mut+ (methanol utilization plus) refers to the wild-type ability of strains to metabolize methanol as the sole carbon source. Mut+ and MutS phenotypes are used when evaluating Pichia transformants for integration of the gene of interest.

Multiple strains are provided so you can test which strains work best for a particular protein. It is important to try at least one Mut+ and one MutS strain.

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.

GS115/pPICZ/lacZ, GS115/Albumin, and GS115 were streaked onto minimal medium containing histidine. Methanol was used as a carbon source. The GS115 and GS115/pPICZ/lacZ grew fast on the methanol plate. The GS115/Albumin grew very slowly on methanol plates. The difference in growth rate confirmed that GS115/pPICZ/lacZ is a Mut+ strain and GS115/Albumin is a MutS strain. The addition of fresh histidine to the plates was critical for this experiment to work.

Proteinase A is a vacuolar aspartyl protease capable of self-activation, as well as subsequent activation of additional vacuolar proteases, such as carboxypeptidase Y and proteinase B. Carobxypeptidase Y appears to be completely inactive prior to proteinase A–mediated proteolytic processing of the enzyme; proteinase B (encoded by the PrB gene of S. cerevisiae) reportedly is approximately 50% bioactive in its precursor form (i.e., the form that exists prior to proteinase A–mediated processing of the enzyme). Little is known about the proteolytic activities in Pichia pastoris. The following protease-deficient Pichia pastoris strains have been made in an attempt to inactivate or delete the homologous proteolytic activities:

SMD 1168: Pep4 gene disrupted 
PichiaPink™ Strain 2: Pep4 gene disrupted 
PichiaPink™ Strain 3: Prb1 gene disrupted 
PichiaPink™ Strain 4: Prb1, Pep4 genes disrupted

The Pep4-deficient mutant is deficient in protease activity of proteinase A, carboxypeptidase Y, and has approximately one-half of proteinase B activity. The Prb1-deficient mutant is deficient in the activity of proteinase B. Finally, the Pep4/PrB–deficient strain is deficient in proteolytic activity of all three of these enzymes: proteinase A, carboxypeptidase Y, and proteinase B. These protease-deficient strains, when compared to protease wild-type Pichia strains, have been shown to be highly efficient expression systems for the production of proteolytically sensitive products. 

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 102 to 103 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 102 to 103 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 103 to 104 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 103 to 104 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.

We recommend electroporation for transformation of Pichia. Electroporation yields 103 to 104 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 Pichia Spheroplast Kit (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. 

The Pichia EasyComp™ Kit produces chemically competent Pichia cells and provides an alternative to electroporation and a rapid, convenient method for transformation. However, because of the low transformation efficiency (3 μg plasmid DNA yields about 50 colonies), it is very difficult to isolate multi-copy integrants. In instances where multi-copy integrants are desired, please use electroporation for best results.

Note: Cells are prepared differently for electroporation. Do not use cells that have been prepared using the EasyComp™ protocol for electroporation.

We strongly recommend electroporation if you are specifically interested in isolating multi-copy integrants of your gene in Pichia. The frequency of multi-copy insertions ranges from 1 to 10%, requiring hundreds to thousands of transformants to isolate a suitable number of multi-copy clones to test for expression. Electroporation yields some of the highest transformation frequencies in Pichia and is the method of choice to isolate multi-copy integrants.

We recommend trying in vivo and in vitro methods to generate or isolate multi-copy inserts of your gene. It is difficult to predict beforehand which method will work better for your protein. A summary of the advantages and disadvantages of each method is presented in the tables below:


                                                                    In Vitro Method (pAO815)

Advantages

Disadvantages

  • Quantitative—construction of a defined number of multimers

 

  • Most of the His+ transformants will contain the proper, defined number of inserts
  • Isolation of recombinants with multiple inserts is easy because most of the His+ transformants will contain multiple copies of the gene of interest

 

  • In vitro construction allows step-wise analysis of copy number effects on protein expression
  • Multiple inserts are located at a single locus

 

  • No need for a second drug resistance marker in the vector
  • More work up front to clone defined number of multimers
  • Size of the vector may become quite large, depending on the size of the gene and the number of copies created

 

  • Rearrangements in E. coli may occur

 

In Vivo Method (pPIC3.5K and pPIC9K)

Advantages

Disadvantages

  • Easy to initiate experiment because only one copy of the gene is cloned into the vector before transforming into Pichia

 

  • Identifies the 1–10% of spontaneous His+ transformants that have multiple inserts
  • Average size of vector is similar to other Pichia expression vectors

 

  • Multiple inserts are located at a single locus

 

  • Qualitative screen–Geneticin® resistance may not necessarily correlate with the number of copies of the gene of interest

 

  • Screening His+ transformants may involve more work because you will need thousands of His+ transformants to generate enough Geneticin® resistant colonies to test
  • The number of multiple inserts is unknown (although this can be determined through Southern or dot blot analysis)

 

  • Screening on Geneticin® is sensitive to the density of the cells and may result in the isolation of false-positives

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

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

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.

  1. Inoculate 10 mL YPD media with Pichia strain and grow O/N, shaking at 30°C.
  2. In the morning, check the OD600. To get them in log phase by the afternoon, dilute cells to hit an OD600 of ~3.0 at 4 or 5 pm.
  3. When the OD600 reaches ~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.
  4. If the OD600 is ~1.0, spin the cells in a 1 L bottle at 3K rpm for 10 minutes.
  5. Gently resuspend in 250 mL cold dH20.
  6. Transfer to a 500 mL centrifuge bottle and spin at 3K for 10 min. Repeat.
  7. Resuspend in 20 mL cold 1 M sorbitol and transfer to a 50 mL conical tube.
  8. Spin at 3K rpm for 10 min.
  9. Resuspend in 1 mL 1M sorbitol, and keep on ice.
  10. Use 80 µL of host strain for each electroporation.

Pichia Fermentation

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

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 chromophore 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 an 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 has a large diameter and long column. Purification of His-tagged proteins on ProBond™ resin would also effectively remove the Aox1p.

The highest pH which still permits growth during Pichia fermentation is 8–8.5.

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 5 L 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.

During the Pichia fermentation process, the nitrogen source is the ammonium hydroxide used to adjust the pH. There is no nitrogen in the basal or trace salts.

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.

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.

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.

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.

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.

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.

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.

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.

Saccharomyces cerevisiae Expression Systems

Here are the different methods available for S. cerevisiae transformation:

  • S. cerevisiae EasyComp™ Transformation Kit: easy-to-use, ready-made reagents
    • Competent cells can be stored frozen. Transformation efficiency is >103 transformants per μg DNA. Higher transformation efficiencies are often obtained with frozen versus freshly prepared cells.
  • Small-scale yeast transformation protocol (page 13 of the manual)
  • Lithium acetate transformation: easy, do-it-yourself protocol
    • Competent cells must be made fresh
  • Electroporation: easy and high efficiency, do-it-yourself protocol
    • Competent cells must be made fresh
  • Spheroplast Kit: high efficiency, a lot of work, not suitable for antibiotic selection

Note: Plate an appropriate density. Colonies will appear over several days. Don’t pick the largest colonies, as these are often suppressors.

 

The efficiency is very strain-dependent, but 1000 to 100,000 transformants per µg DNA is the range.

Stock buffers of TE, lithium acetate, and PEG can be stored. However, the combined solution 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.

We offer the INVSc1 yeast strain. It is a diploid strain for expression purposes only. It does not sporulate well and is therefore not suited for yeast genetic studies. The genotype and phenotype of the INVSc1 strain are as follows:

Genotype: MATa his3Δ1 leu2 trp1-289 ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-52

Phenotype: His-, Leu-, Trp-, Ura-

Note that INVSc1 is auxotrophic for histidine, leucine, tryptophan, and uracil. The strain will not grow in SC minimal medium that is deficient in histidine, leucine, tryptophan, and uracil.

The trp1-289 mutation is a point mutation in the TRP-1 gene that causes this strain to be auxotrophic for tryptophan (i.e., tryptophan is required in growth media for this strain). There are many vectors that contain a wild-type copy of the TRP1 gene that will complement the trp1-289 mutant phenotype and can therefore be used as a selectable marker for such vectors. The phenotype of a trp1-289 mutant and a trp1 complete deletion mutant are similar, but strains with the trp1 complete deletion, unlike trp1-289, will not induce well with galactose. Therefore, when galactose induction is used, it is better to use a trp1-289 mutant. However, since trp1-289 mutants revert with a detectable frequency, it is important to verify your clones.

Some researchers choose to grow yeast in medium containing 2% galactose as the sole carbon source during induction. However, yeast typically grow more quickly in induction medium containing 2% galactose plus 2% raffinose. Raffinose is a good carbon source for yeast, and unlike glucose, does not repress transcription from the GAL promoter. Raffinose is a trisaccharide of galactose, glucose, and fructose linked in that order. Most yeast can cleave the glucose-fructose bond, but not the galactose-glucose bond. Fructose is then used as a carbon source.

pYES-DEST52 (Cat. No. 12286019) is the only yeast expression vector we offer that is Gateway-compatible. It is designed for high-level, galactose-inducible expression in Saccharomyces cerevisiae.

Yes, we do offer the pYES2.1/V5-His-TOPO® vector, which is part of the pYES2.1 TOPO® TA Expression Kit (Cat. No. K415001), for the direct cloning of Taq polymerase–amplified PCR products and regulated expression in Saccharomyces cerevisiae using galactose.

S. pombe cannot generate P factor when P factor is replaced for alpha in the alpha factor gene. It can, however, produce alpha factor when alpha is replaced for P in the P factor gene. This is negative evidence that S. pombe can process its own mating factor cleavage sites, but not all the cleavage sites of the S. cerevisiae alpha factor. It is better to use a more generic signal sequence (rather than a pre- pro- signal sequence such as alpha). If it is necessary to go the pre- pro- route, it is better to use the S. pombe P factor leader rather than the S. cerevisiae alpha leader.

Yeast Growth Media

Following are the rich and minimal media used for culturing Pichia pastoris and S. cerevisiae:

Rich Media:

S. cerevisiae and Pichia pastoris

  • YPD (YEPD): yeast extract, peptone, and dextrose
  • YPDS: yeast extract, peptone, dextrose, and sorbitol

Pichia pastoris only

  • BMGY: buffered glycerol-complex medium
  • BMMY: buffered methanol-complex medium

Minimal Media (also known as drop-out media):

S. cerevisiae

  • SC (SD): Synthetic complete (YNB, dextrose (or raffinose or galactose), and amino acids)

Pichia pastoris

  • MGY: minimal glycerol medium
  • MD: minimal dextrose
  • MM: minimal methanol
  • BMGH: buffered minimal glycerol
  • BMMH: buffered minimal methanol

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

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

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.