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Cat. No.: PTPP-50 (for 50T)

Cat. No.: PTPP-250 (for 250T)

Description

Competent Cell Preparation and Transformation Kit for Pichia Pastoris is a rapid, convenient, and efficient kit for preparing Pichia pastoris competent cells and performing plasmid transformation. During Pichia pastoris transformation, yeast cells are cultured in the buffer solution provided in the kit to prepare them for transformation. Then, competent cells are mixed with the plasmid DNA to be transformed and Carrier DNA, and transformation is carried out by incubating the mixture with the transformation solution. Carrier DNA is short single-stranded linear DNA that facilitates the entry of plasmids into yeast cells during the uptake of exogenous plasmid DNA, and it also protects plasmids from degradation by DNA enzymes.

Pichia pastoris has various advantages in its eukaryotic expression system, including good post-translational protein processing, protein folding, and post-translational modifications. Additionally, it shares ease of operation similar to Escherichia coli and Saccharomyces cerevisiae. Compared to insect cell expression systems and mammalian cell expression systems, the Pichia pastoris expression system is rapid, efficient, and economical, with foreign proteins typically exhibiting high expression levels. Compared to Saccharomyces cerevisiae, Pichia pastoris often exhibits expression levels of foreign proteins that are 10 to 100 times higher and can perform N-linked glycosylation modifications on secreted proteins. However, there are differences in the length of glycan chains when expressing and modifying secreted proteins. Typically, Pichia pastoris-modified secreted proteins have glycan chain lengths of 8-14 mannose residues, whereas Saccharomyces cerevisiae-modified secreted proteins have glycan chain lengths of 50-150 mannose residues. Additionally, Pichia pastoris rarely performs O-linked glycosylation modifications on secreted proteins.

Unlike hosts such as Escherichia coli and Saccharomyces cerevisiae, Pichia pastoris itself does not have stably existing free plasmids. All expression vectors must be integrated into the yeast genome, meaning Pichia pastoris transformation results in the generation of genetically stable transformants by integrating plasmid DNA into specific sites in the yeast genome. Common Pichia pastoris expression vectors usually contain the HIS4 marker gene. These vectors, when linearized by restriction enzymes, undergo recombination at the AOX1 locus or his4 locus to produce His+ recombinants.

Homologous recombination occurs during Pichia pastoris plasmid transformation, requiring homologous arms at both ends of the target gene. Circular plasmids, once cut at specific sites, have yeast homologous sequences at both ends of the entire expression cassette. Thus, they can be integrated into the yeast genome through homologous recombination and selected via the resistance gene expressed in the expression cassette. In fact, even if the vector is not linearized by enzymatic digestion during transformation, it can still be integrated into the yeast genome, but the efficiency of recombination is low. Linearization significantly enhances the efficiency of homologous recombination at specific sites. When selecting linearization sites, ensure that the target gene inserted does not contain restriction enzyme recognition sites intended for linearization. Different types of transformants are produced depending on the linearization site, as shown in the table below. The linearization site of the vector often determines the phenotype of the transformants. When transformed into Pichia pastoris strain GS115, two types of transformants, His+Mut+ and His+Muts, are produced, while in strain KM71, only His+Muts transformants are produced due to the Muts phenotype of this strain.

Components

• Buffer A
• Buffer B
• Buffer C
• Carrier DNA
• DMSO

Storage

Store at -20ºC, effective for at least one year. Except for Carrier DNA, store at 4ºC, effective for one year.

Precaution

• When using Carrier DNA, heat the required amount of Carrier DNA at 100ºC for 15 minutes according to the transformation quantity, then immediately place it on ice. After 15 minutes, it can be used. Store it at -20ºC after use. Thaw the Carrier DNA on ice and reboil to denature before next use. If the amount used each time is small, it can be properly aliquoted to avoid repeated freeze-thaw cycles.
• The volume of the sample to be transformed should not be too large, as a large volume may lead to decreased transformation efficiency.
• Competent cells are very sensitive to temperature changes, so avoid temperature changes beyond the usage instructions.
• Competent cells are sensitive to mechanical force. When adding the sample to be transformed, handle it gently, and avoid vigorous mixing with a pipette.
• It is generally recommended to take a portion of the sample for transformation. In case of transformation failure, there will still be a portion of the sample available for repeat transformation.
• All components of this kit have been sterilized. Please operate in a clean bench when in use and seal it after use to avoid contamination.
• This product is intended for scientific research use by professionals only and is not intended for clinical diagnosis or treatment, food, or drug purposes. It should not be stored in residential settings.
• For your safety and health, wear laboratory coats and disposable gloves when handling.

Related:

• Yeast Colony PCR Kit (Alkaline Lysis)
• Yeast Colony PCR Kit (Enzymatic Lysis)
• Competent Cell Preparation and Transformation Kit for Pichia Pastoris
• Competent Cell Preparation and Transformation Kit for Saccharomyces Cerevisiae

Only for research and not intended for treatment of humans or animals

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