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货号 | 数量 |
---|---|
V19020 | 20 μg |
The Pichia Expression System (a.k.a., the Pichia Classic Expression System, see https://pichia.com/classic-pichia/) is based on strains of Pichia pastoris yeast and corresponding genetic expression vectors. Pichia pastoris expresses most recombinant proteins and other substances at high levels. All strains and vectors for the Pichia Expression System are owned or controlled by Research Corporation Technologies, Inc., Tucson, Arizona (“RCT”). Thermo Fisher Scientific Inc. (“Thermo Fisher”) is the exclusive distributor of kits containing various combinations of RCT’s Pichia pastoris strains and vectors (each a “Kit”) with the right to extend the license granted hereunder (the “Limited Use Label License” or “LUL”). Each Kit may contain one or more of the following:
Strains | Vectors |
---|---|
GS115 | pHIL-D2 |
X-33 | pPIC3K |
KM71 | pPIC3.5K |
KM71H | pPIC9K |
SMD1168 | pA0815 |
SMD1168H | pPICZ A, B, & C pPICZalpha A, B, & C pPIC6Zalpha A, B, & C pGAPZ A, B, & C pGAPZalpha A, B, & C |
You may use the Kit materials only for research purposes, as further described below. You may not use the Kit materials for any commercial purpose without first obtaining a commercial license from RCT, as detailed below.
Your use of the strains and vectors listed above (“Kit Materials”) is conditioned on your agreeing to this LULL. If you do not agree to this LULL, you must contact Thermo Fisher within 10 days after the date of purchase for authorization to return the unopened and unused Kit for a full refund. If you do not timely contact Thermo Fisher, you will be presumed to have agreed to the terms of this LULL and to be bound by it and will not be able to obtain a refund.
Thermo Fisher grants you a non-exclusive, revocable, limited, non-transferable license to use the Kit Materials and Kit Information to produce and use Expression Product (as defined below) solely for non-commercial research or internal evaluation purposes. You may not sequence any of the Kit Materials or otherwise reverse engineer them. You may not use the Kit Materials or Kit Information for any Commercial Purpose (as defined below) without a license for such purpose from RCT. RCT may contact you during the term of this LULL regarding your desire for a commercial license.
“Commercial Entity” means any entity engaged in commercial activities for a profit or income.
“Commercial Purpose” means: (1) any use of Kit Materials, Kit Information, or Expression Product(s) in a Commercial Product (as defined below); (2) any use of Kit Materials, Kit Information, or Expression Product(s) in the manufacture of a Commercial Product; (3) any use of Kit Materials, Kit Information, or Expression Product(s) in performing services for any third party; (4) any sale or other commercialization of Expression Product(s); (5) any use of Kit Materials, Kit Information, or Expression Product(s) directly or indirectly in the clinical development, improvement, or GMP manufacture of a Commercial Product; and (6) the filing or preparation of any patent application claiming any Kit Materials or any derivatives of the Kit Materials.
“Commercial Product” means any product intended for sale or use for a Commercial Purpose.
“Derivative” means any strain (including any transformed strain) or vector that is derived, in whole or in part, from any Kit Materials or using any Kit Information, or that is the progeny of any Kit Materials. “Derivative” excludes genes and genetic components that are proprietary to you and have not been obtained through use of the Kit Materials or Kit Information. “Expression Product” means any substance, protein, peptide, polypeptide, or nucleic acid expressed with the use of one or more Kit Materials and any Derivatives. “Research Institution” means a non-profit entity that has, as its principal purposes, education and the conduct of research to provide a benefit to the public and, with respect to the Kit Materials, Derivatives, or Kit Information, is not engaged in any commercial activities for profit or income.
You must limit access to any Kit and the Kit Materials solely to your officers, employees, or agents (and, in the case of Research Institutions, your students) who need access to perform the research or evaluation for which the Kit was purchased. You must advise each recipient of Kit Materials that the Kit Materials must be used subject to this LULL and require each recipient to agree, in writing, to be bound by this LULL. The Kit Materials and all Derivatives are owned or controlled by RCT, and you and each user hereby assigns, and agrees to assign, to RCT all right, title and interest in and to any Derivatives. You may not distribute any Kit Materials or Derivatives to persons, including without limitation those within your own organization, without RCT’s prior written consent, which may be withheld in RCT’s sole discretion. You may not assign, sublicense, or otherwise transfer this LULL or any right, license, or obligation under this LULL without RCT’s prior written consent.
Commercial Entities may conduct their non-commercial research and internal evaluation for one year from date of purchase at which time this LULL automatically terminates. Commercial entities will be contacted by RCT during the evaluation period to explore your interest in obtaining a commercial license or research license to allow continued use of the Kit Materials and Kit Information. Research Institutions may conduct their non-commercial research and evaluation for so long as each Research Institution is in compliance with the terms of this LULL, and if not, this LULL automatically terminates.
You may terminate this LULL at any time by ceasing any and all use of any and all Kit Materials, Derivatives, and Expression Products. This LULL also terminates automatically and immediately if you fail to comply with it in any respect. Upon expiration or termination of this LULL, you must immediately: (a) cease any and all use of all Kits, Kit Materials, Derivatives, and any and all Expression Products; and (b) provide RCT written or electronic notification confirming cessation of any and all use. In its sole discretion, RCT may require you to destroy all your Kit(s), Kit Materials, and Derivatives.
RCT is an express and intended third-party beneficiary of this LULL and may independently enforce its terms and provisions in its sole and absolute discretion. You may contact Research Corporation Technologies at the following address: Brad Morie, Senior Director, Research Corporation Technologies, Inc. 6440 N. Swan Road, Suite 200, Tucson, AZ 85718, (520) 748-4413 (o), (520) 748-0025 (f), bmorie@rctech.com.
(Rev 7/2024)
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.
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.
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.
Following are the rich and minimal media used for culturing Pichia pastoris and S. cerevisiae:
Rich Media:
S. cerevisiae and Pichia pastoris
YPD (YEPD): yeast extract, peptone, and dextrose
YPDS: yeast extract, peptone, dextrose, and sorbitol
Pichia pastoris only
BMGY: buffered glycerol-complex medium
BMMY: buffered methanol-complex medium
Minimal Media (also known as drop-out media):
S. cerevisiae
SC (SD): Synthetic complete (YNB, dextrose (or raffinose or galactose), and amino acids)
Pichia pastoris
MGY: minimal glycerol medium
MD: minimal dextrose
MM: minimal methanol
BMGH: buffered minimal glycerol
BMMH: buffered minimal methanol
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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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