PichiaPink™ Secreted Vector Kit
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Invitrogen™

PichiaPink™ Secreted Vector Kit

The PichiaPink™ Secreted Vector Kit contains the pPINKα-HC vector for use with the PichiaPink™ Yeast Expression System. The pPINKα-HC vectorRead more
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Catalog NumberQuantity
A111531 kit
Catalog number A11153
Price (CNY)
20,841.00
Each
Add to cart
Quantity:
1 kit
Price (CNY)
20,841.00
Each
Add to cart
The PichiaPink™ Secreted Vector Kit contains the pPINKα-HC vector for use with the PichiaPink™ Yeast Expression System. The pPINKα-HC vector is built around the ADE2 gene for complementing the complementing the ade2 deletion in the PichiaPink™ strains. The pPINKα-HC vector has the α-mating factor secretion signal sequence so that all proteins are secreted. You can order the PichiaPink™ Secreted Vector Kit to refill your PichiaPink™ Yeast Expression System Kit.

The PichiaPink™ Secreted Vector Kit comes with:
•     pPINKα-HC vector (high-copy number) (20 μg)
•     5’α-factor sequencing primer (20 μg)
•     3’CYC1 sequencing primer (20 μg)
•     One Shot™ TOP10™ Electrocomp™ E. coli (Cat. No. C404052)

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.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Related Links
• Download the pPINKα-HC plasmid map (PDF).
• Learn more about the PichiaPink™ Yeast Expression System.
• Learn more about our other protein expression systems.

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
Antibiotic Resistance BacterialAmpicillin (AmpR)
CleavageNo Cleavage Site
FormatLiquid
Inducing AgentMethanol
Product TypeVector Kit
Quantity1 kit
Replication OriginpUC Origin
Shipping ConditionRoom Temperature
VectorpPink
Cloning MethodRestriction Enzyme/MCS
PromoterAOX1
Protein TagUntagged
Unit SizeEach
Contents & Storage
PichiaPink™ Secreted Protein Vector kit contains 20 μg pPinkα-HC (the high-copy number plasmid with the S. cerevisiae Alpha mating factor presequence) with sequencing primers 5' α-factor and 3' CYC1. Store at -20°C.

Frequently asked questions (FAQs)

I am using the pPink-HC vector and the transformation efficiency of the host strain seems to be very low. Why is this?

When using the pPink-HC vector, the transformation efficiency of the host strain may appear low because colonies with only a few copies of the marker will not produce enough ADE2 gene product to grow and will be selected against on medium lacking adenine (i.e., adenine dropout medium or minimal medium). Only colonies that have integrated multiple copies of the ADE2 marker will able to grow without adenine. Since the gene of interest is linked to the selection marker, the white colonies could also result in higher expression of the gene of interest.

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.

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