Why are protease deficient Pichia pastoris and Pichia methanolica strains used for protein expression? What strains are available?

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DESCRIPTION OF PROTEOLYTIC ACTIVITIES
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
SMD 1165 PrB gene disrupted
SMD 1163 Pep4/PrB gene disrupted
PichiaPink™ Strain 2 Pep4 gene disrupted
PichiaPink™ Strain 3 Prb1 gene disrupted
PichiaPink™ Strain 4 Prb1, Pep4 gene disrupted

The Pep4 deficient mutant theoretically reduces the protease activity of Proteinase A, Caboxypeptisae Y, and approximately one-half of Proteinase B activity. The proteinase B deficient strain only reduces the activity of proteinase B. Finally, the Pep4/PrB strain reduces or eliminates the proteolytic activity of all three of these enzymes, proteinase A, Carboxypeptidase Y and Proteinase B. These protease deficient strains when compared to wild-type Pichia strains have shown to be highly efficient expression systems for the production of proteolytically sensitive products.

The PRB1 deficient mutant is deficient in expression of proteinase B.

PREPARATION OF PROTEASE DEFICIENT STRAINS
The preferred method for preparing Pichia strains deficient in proteolytic activity, specific disruption of protease-encoding genes, was achieved by gene addition, gene replacement or a combination of additions and replacement referred to as "pop-in-pop-out" method. In gene replacement, the endogenous target gene is physically removed from the target locus, and replaced with a modified gene. This a accomplished by transforming the host with a linear fragment having ends which are homologous to the 5' and 3' ends of the target gene respectively. Gene addition involves adding the transforming DNA to the endogenous target gene. Depending on the manner in which the modified gene of the transforming DNA was altered, gene addition can result in the presence of either two non-functional copies of the target gene, or one functional and one non-functional copy of the target gene. Each of the two copies consists of a portion of the transforming DNA. If a functional copy of the target gene remains after gene addition, it can be removed by homologous recombination between the two copies of the target gene. The combination process of gene addition followed by homologous recombination constitutes the "Pop-in-pop-out" process.

USEFULNESS
When proteolytically sensitive recombinant products such as epidermal growth factor (EGF), growth hormone releasing factor (GRF), Insulin-like growth factor-1 (IGF-1), are expressed in Pichia strains which are deficient in proteolytic activity, higher levels of authentic bioactive recombinant product are produced. The following example illustrates the usefulness of these protease deficient strains:

Proteolytic activity of the broth from a normal Pichia strain and a Pep4- Pichia strain were compared by adding known amount of peptide to the broth. The stability of the peptides in the two different broths was monitored by HPLC; when GRF or EFG were incubated with cell-free broth from these two strains, less than 10% of the peptides remained intact after 4 hours. In contrast, the GRF was greater than 60% intact after 4 hours and EGF remained greater than 90% after 8 hours incubation in the cell-free broth of the Pep4- strain. These data demonstrate that the disruption of the pep4 gene of Pichia result in a substantial reduction of the proteolyis.

Answer Id: E3769

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4c764e970467a46dd07d77a4c7d671a1_FAQ

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

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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

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144aa13d4ee5859d027db8b714afb8c8_FAQ

What selection mechanism does the PichiaPink™ Yeast Expression System use?

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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.

Answer Id: E9482

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d6645e693dd2e1709a153f64cf3b6164_FAQ

What is the size of the Pichia genomic DNA?

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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

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f1c7dffa78cf7301e26fcfdc09501727_FAQ

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

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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

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950da299ce3cb0f447cae0ffb31f8590_FAQ

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

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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).

Answer Id: E9490

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4c424a46388eef35830f97f0898766f6_FAQ

What is the codon usage for Pichia?

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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

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7f29bfefc9ba6219545676bbafd4ffa1_FAQ

Will a mammalian secretion signal work in Pichia?

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

Answer Id: E9494

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61d33ea522645ed9dd8e8d1ff55e9adc_FAQ

How is the alpha factor secretion signal sequence processed?

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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.

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5a693d86c2746d38c3dadf57eccbfd35_FAQ

Will the alpha factor secretion signal work in other yeast?

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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

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52c3fa36c9c325ea42391226a306d4e5_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?

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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

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14d2754c6570f402a556e3b8f668da61_FAQ

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

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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

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2caa83fb8d84ca41e15d5c534d10292f_FAQ

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

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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.

Answer Id: E9502

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0129767d890b21eed19766627406dacb_FAQ

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

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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.

Answer Id: E9503

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3aa06b5761d353ecefc474cfb3a0f1e6_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?

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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

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72f19698f6205dc418b1f6bdc8ea1a90_FAQ