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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How can I freeze Pichia?

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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: E4227

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

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Here are some suggestinos:

- Make sure that you have harvested cells during log-phase growth (OD <1.0 generally).
- If electroporation is being used, see the electroporator manual for suggested conditions. Vary electroporation parameters if necessary.
- Use more DNA.
- Use freshly made competent cells.
- If the LiCl transformation method is being used, try boiling the carrier DNA.

Answer Id: E9561

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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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Can YPD be used instead of BMGY-type media for pichia fermentation?

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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. The richer media makes it difficult to purify secreted proteins from the media. The BMGY formulation remedies both of these problems.

Answer Id: E3755

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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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When selecting for blasticidin-resistant transformants in the X-33 strain using pPIC6/pPIC6alpha vectors, why do I get large and small colonies on YPD plates containing 300 μg/ml blasticidin?

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Generally, large colonies represent transformants containing pPIC6/pPIC6alpha integrants, while small colonies represent transformants containing pPIC6/pPIC6alpha non-integrants. These non-integrants have transduced the pPIC6/pPIC6alpha plasmid, and therefore, exhibit a low level of blasticidin resistance in the initial selection process. Upon subsequent screening, these non-integrant transformants do not retain blasticidin resistance.

When choosing a blasticidin-resistant transformant for your expression studies, we recommend that you pick blasticidin-resistant colonies from the initial transformation plate and streak them on a second YPD plate containing the appropriate concentration of blasticidin. Select transformants that remain blasticidin-resistant for further studies.

Answer Id: E9562

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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.

Answer Id: E9495

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Why would I pick a yeast expression system for expression of my protein, as opposed to expression systems in other hosts?

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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.

Answer Id: E9477

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Do you need to add sulfuric acid to the Fermentation PTM Trace salts?

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You don't have to add sulfuric acid to your PTM1 salts or fermentation media. It would serve no purpose, other than may be help dissolve the salts.

Answer Id: E3756

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

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

Answer Id: E9531

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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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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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My spheroplasting of Pichia worked twice, but hasn't worked since. The OD of the culture simply does not drop.

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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

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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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