Citations et références (4)

Invitrogen™

Vecteurs pPICZα A, B, & C Pichia

Les vecteurs pPICα A, B et C sont des vecteurs de 3,6 kb utilisés pour exprimer et sécréter des protéinesAfficher plus

Référence	Quantité
V19520	20 μg

Référence V19520

Prix (EUR)

1 354,00

20 µg

Quantité:

20 μg

Prix (EUR)

1 354,00

20 µg

Les vecteurs pPICα A, B et C sont des vecteurs de 3,6 kb utilisés pour exprimer et sécréter des protéines recombinantes dans Pichia pastoris. Les protéines recombinantes sont exprimées en fusions avec un peptide d’extrémité N-terminale codant le signal de sécrétion du facteur á de Saccharomyces cerevisiae. Ces vecteurs permettent une expression inductible par le méthanol de haut niveau du gène d’intérêt dans Pichia et peuvent être utilisés dans n’importe quelle souche Pichia, y compris X-33, SMD1168H et KM71H. Les vecteurs pPICα contiennent les éléments suivants :

• Contient le promoteur AOX1 pour l’expression du gène d’intérêt induite par méthanol et étroitement régulée
• Les trois cadres de lecture (versions A, B, C) sont fournis pour faciliter le clonage à l’intérieur du cadre avec le peptide d’extrémité C-terminale
• Signal de sécrétion du facteur α pour diriger l’expression sécrétée de protéines recombinantes
• Gène de résistance à la Zeocin pour une sélection à la fois dans E. coli et Pichia
• Peptide d’extrémité C-terminale contenant l’épitope c-myc et un marqueur de polyhistidine (6xHis) pour la détection et la purification de protéines de fusion recombinantes

Usage exclusivement réservé à la recherche. Ne pas utiliser pour des procédures de diagnostic.

Spécifications

Résistance aux antibiotiques des bactériesZeocin™ (ZeoR)

Type de produitVecteur d’expression Pichia

Quantité20 μg

Conditions d’expéditionTempérature ambiante

VecteurpPIC

Méthode de clonageEnzyme de restriction / MCS

AccélérateurAOX1

Marqueur de protéineMarqueur His (6x), marqueur d’épitope c-Myc

Unit Size20 µg

Contenu et stockage

20 µg chacun de PICZ lyophilisé A, B et C est fourni (stockage à -20°C)

Foire aux questions (FAQ)

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.

What are the different kinds of media used for culturing Pichia pastoris and S. cerevisiae?

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.

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.

View all Vecteurs pPICZα A, B, & C Pichia FAQs

Citations et références (4)

Citations et références

Abstract

Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris.

Authors:FitzGerald K, Holliger P, Winter G,

Journal:Protein Eng

PubMed ID:9488147

'Diabodies are dimeric antibody fragments held together by associated heavy and light chain variable domains present on different polypeptide chains. To improve their stability we have introduced cysteine residues into the V-domains to promote the disulphide crosslinking of the dimer. A crosslinked bivalent diabody against carcinoembryonic antigen (CEA) and a ... More

Distinct kinetics for binding of the CD46 and SLAM receptors to overlapping sites in the measles virus hemagglutinin protein.

Journal:J Biol Chem

PubMed ID:12065582

Measles virus (MV) is a human pathogen using two distinct cell surface receptors for entry into host cells. We present here a comparative analysis for binding of the MV receptors CD46 and SLAM to the measles virus hemagglutinin protein (MVH, Edmonston strain). Soluble monomeric and dimeric MVH variants were prepared ... More

Insertional mutagenesis and immunochemical analysis of visual arrestin interaction with rhodopsin.

Authors: Dinculescu Astra; McDowell J Hugh; Amici Stephanie A; Dugger Donald R; Richards Nigel; Hargrave Paul A; Smith W Clay;

Journal:J Biol Chem

PubMed ID:11809770

Visual arrestin inactivates the phototransduction cascade by specifically binding to light-activated phosphorylated rhodopsin. This study describes the combined use of insertional mutagenesis and immunochemical approaches to probe the structural determinants of arrestin function. Recombinant arrestins with insertions of a 10-amino acid c-Myc tag (EQKLISEEDL) were expressed in yeast and characterized. ... More

Yeast Expression and NMR Analysis of the Extracellular Domain of Muscle Nicotinic Acetylcholine Receptor alpha Subunit.

Authors: Yao Yun; Wang Junmei; Viroonchatapan Nitnara; Samson Avraham; Chill Jordan; Rothe Elizabeth; Anglister Jacob; Wang Zuo-Zhong;

Journal:J Biol Chem

PubMed ID:11812776

The alpha subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for alpha-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of ... More

Vecteurs pPICZα A, B, & C Pichia

Foire aux questions (FAQ)

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

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

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

What are the different kinds of media used for culturing Pichia pastoris and S. cerevisiae?

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

Citations et références (4)

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?