PichiaPink™ Secretion Optimization Kit
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PichiaPink™ Secretion Optimization Kit

The PichiaPink™ Secretion Optimization Kit is a new recombinant protein expression system based on the yeast Pichia pastoris. The PichiaPink™深入閱讀
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產品號碼Quantity
A111501 Kit
產品號碼 A11150
價格 (HKD)
-
Quantity:
1 Kit

The PichiaPink™ Secretion Optimization Kit is a new recombinant protein expression system based on the yeast Pichia pastoris. The PichiaPink™ Secretion Optimization Kit gives you convenient and cost-efficient bioproduction from shake flask to 1000-liter production of your recombinant protein. The PichiaPink™ Secretion Optimization Kit comes with our new Pichia strains, the pPINK-HC and pPINK-LC vectors, and secretion signal sequences for production of your protein.

  • Get a complete kit to get started with protein production in Pichia pastoris.
  • Screen your Pichia transformants rapidly by auxotrophy and color selection.
  • Reduce protein degradation with protease-deficient Pichia pastoris strains.
  • Optimize protein secretion with one of 8 secretion signal sequences.

The PichiaPink™ Secretion Optimization Kit comes with:

  • PichiaPink™ Vector Kit (Cat. No. A11152)
  • PichiaPink™ Secretion Signal Set (A11155)
  • PichiaPink™ Expression Strain Set (A11154)
  • PichiaPink™ Media Kit (A11156)

A New Way to Screen Pichia Transformants

The PichiaPink™ Secretion Optimization Kit uses adenine auxotrophy to select for Pichia transformants. The PichiaPink™ strains have the ade2 genotype and require complementation with the ADE2 gene to grow without adenine. Use of an auxotrophic marker also makes it easier to maintain transformants. No antibiotics are required to maintain your transformants.

In addition, you can screen for Pichia transformants by color. The untransformed PichiaPink™ strains appear as pink colonies while transformed PichiaPink™ strains appear as white colonies (Fig 1).

New Expression Vectors for Pichia

The PichiaPink™ Secretion Optimization Kit comes with the new PichiaPink™ Vector Kit. The kit includes two vectors, pPINK-LC and pPINK-HC. The pPINK vectors are built around the ADE2 gene for complementing the ade2-deficient Pichia strains. Both vectors use the methanol-induced AOX1 promoter to express your protein. The pPINK-LC integrates at low-copy number into the Pichia genome while the pPINK-HC vector integrates with high-copy number.

Both pPINK-LC and pPINK-HC vectors are compatible with the PichiaPink™ Secretion Signal Set. Using a 3-way ligation, you can add one of 8 secretion sequences to your gene. Note: the pPINK vectors express proteins intracellularly by default.

Both the PichiaPink™ Vector Kit (Cat. No. A11152) and PichiaPink™ Secretion Signal Set (A11155) can be ordered separately to refill the PichiaPink™ Secretion Optimization Kit.

Keep Your Proteins Intact

The PichiaPink™ Secretion Optimization Kit comes with the PichiaPink™ Expression Strain Set. This set contains the 4 Pichia pastoris strains for use with the PichiaPink™ Expression System. All 4 strains have the ade2 genotype. In addition, 3 strains include knockouts in 2 protease genes. Protease-deficient strains reduce the need for protease inhibitors when growing your cells and improve the yield of your protein. Test a single protease knockout or the double protease knockout to find the one that works best for you.

You can order the PichiaPink™ Expression Strain Set (Cat. No. A11154) separately to refill the PichiaPink™ Secretion Optimization Kit.

Media Pouches to Get You Started

The PichiaPink™ Media Kit is a set of convenient prepackaged media pouches for use with the PichiaPink™ Secretion Optimization Kit. The PichiaPink™ Media Kit contains media needed to grow PichiaPink™ strains from frozen stocks to cultures ready for transformation and selection. You can order the PichiaPink™ Media Kit (Cat. No. A11156) separately to refill the PichiaPink™ Secretion Optimization Kit.

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.
規格
Bacterial or Yeast StrainPichiaPink™
Expression MechanismCell-Based Expression
Expression StabilityStable
Expression SystemYeast
For Use With (Application)Protein Expression
Key FunctionsSecreted Expression
Product TypeOptimization Kit
Quantity1 Kit
Shipping ConditionRoom Temperature
Cell TypeYeast Cells
FormatKit
PromoterAOX1
SpeciesP. pastoris
VectorpPINK-LC, pPINK-HC
Unit SizeEach
內容物與存放
The PichiaPink™ Secretion Optimization Kit consists of:
• PichiaPink™ Vector kit containing a high- and a low copy number plasmid
• PichiaPink™ Secretion Signal set containing 8 secretion signals
• PichiaPink™ Expression Strain set containing 4 glycerol strains
• PichiaPink™ Media Kit containing 4 basic media

Store vectors and signal sequences at -20°C
Store the PichiaPicha™ strains at -80°C
Store the Media Kit at room temperature

常見問答集 (常見問題)

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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