PichiaPink™ Secretion Signal Set
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PichiaPink™ Secretion Signal Set

The PichiaPink™ Secretion Signal Set is a set of 8 DNA fragments encoding secretion signal sequences. The PichiaPink™ Secretion SignalRead more
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Catalog number A11155
Price (USD)
2,980.00
Each
Add to cart
Price (USD)
2,980.00
Each
Add to cart

The PichiaPink™ Secretion Signal Set is a set of 8 DNA fragments encoding secretion signal sequences. The PichiaPink™ Secretion Signal Set is designed to be easily ligated in front of your gene. The PichiaPink™ Secretion Signal Set is designed for use with the pPINK-HC and pPINK-LC vectors.

Optimize Secretion of Your Expressed Protein

By adding one of the 8 secretion signal sequences to your gene, you can secrete your protein into the media to make it easier to harvest and purify. Pichia pastoris secretes few native proteins, so the bulk of the protein in the media will be yours.

Each DNA sequence encodes a different secretion signal. You can test each secretion signal sequence to find the one that works best for your protein.

The PichiaPink™ Secretion Signal Set comes with 8 different secretion signals including:

  • α-mating factor pre-sequence from Saccharomyces cerevisiae
  • α-amylase signal sequence from Aspergillus niger
  • Glucoamylase signal sequence from Aspergillus awamori
  • Serum albumin signal sequence from Homo sapiens
  • Inulinase signal sequence from Kluyveromcyes maxianus
  • Invertase signal sequence from Saccharomyces cerevisiae
  • Killer protein signal sequence from Saccharomyces cerevisiae
  • Lysozyme signal sequence from Gallus gallus

Easily Add a Secretion Signal to Your Protein

The DNA fragments in the PichiaPink™ Secretion Signal Set are designed to be easily inserted in front of your gene when using the pPINK-HC or pPINK-LC vectors. You can try different secretion signal sequences to see which one gives optimal secretion of your protein.

Each signal sequence includes the Kozak sequence for the AOX1 gene from Pichia pastoris to ensure efficient translation of your protein.

The secretion signal sequences come as phosphorylated duplex oligomers in 40 pmol aliquots. Resuspend oligomers in 40 μL TE buffer before use.

Why Choose the PichiaPink™ Yeast Expression System?

The PichiaPink™ Yeast Expression System is based on the yeast Pichia pastoris. Advantages of Pichia pastoris include rapid growth, well-defined genetic background, simple media formulation, and easy handling. For over 30 years, Pichia pastoris has been used by labs around the world for producing hundreds of different proteins from many species including human (Ref 1, 2). The PichiaPink™ Yeast Expression System allows convenient and cost-effective protein production from small to large scales.

For information on obtaining a commercial-use license for the PichiaPink™Yeast Expression System, please inquire at outlicensing@lifetech.com.

References

1. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45-66. [PubMed]

2. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol. 2002 Aug;13(4):329-32. [PubMed]

For Research Use Only. Not for use in diagnostic procedures.
Specifications
Bacterial or Yeast StrainPichiaPink™
Cloning MethodRestriction Enzyme/MCS
DescriptionCloning into expression vectors
PromoterAOX1
SpeciesP. pastoris
VectorpPINK-LC, pPINK-HC
Expression MechanismCell-Based Expression
For Use With (Application)Protein Expression
Product TypeSecretion Signal Set
Quantity1 Kit
Shipping ConditionRoom Temperature
Unit SizeEach
Contents & Storage
The PichiaPink™ Secretion Signal Set consists of 8 signal sequences which are DNA duplexes:
• S. cerevisiae Alpha mating factor presequence
• Asp. niger alpha Amylase
• Asp. awarmori Glucoamylase signal
• H. sapien serum albumin signal
• K. maxianus Inulinase presequence
• S.cerevisiae Killer Protein sequence
• S.Cerevisiae invertase Signal sequence
• G. gallus Lysozyme Signal sequence

Quantity: 40 pMoles
Store at -20°C.

Frequently asked questions (FAQs)

When selecting for blasticidin-resistant transformants in the X-33 strain using pPIC6/pPIC6α vectors, why do I get large and small colonies on YPD plates containing 300 µg/ml blasticidin?

Generally, large colonies represent transformants containing pPIC6/pPIC6α integrants, while small colonies represent transformants containing pPIC6/pPIC6α non-integrants. These non-integrants have transduced the pPIC6/pPIC6α 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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:

- If the OD of cells that are used is too high, they will not spheroplast. Do not overgrow cells.
- Do not use old cells and make sure that they are in log phase of growth.
- Make sure to mix zymolyase well before using. Zymolyase is more of a suspension than a solution.
- Make the PEG solution fresh each time and check the pH.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Is there a recommended protocol for fermentation using constitutive expression vectors such as pGAPZ?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can the methanol and ammonium hydroxide solutions used to prepare Pichia fermentation medium 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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