PichiaPink-2 Chemically Competent Cell

PichiaPink-2 Chemically Competent Cell

PichiaPink-2 is a Pink strain of Pichia pastoris, belonging to eukaryotic cells. Common antibiotics targeting prokaryotic organisms, such as kanamycin and ampicillin, are ineffective against yeast, so to prevent contamination of yeast cultures by prokaryotic organisms like E. coli, antibiotics such as ampicillin and kanamycin are often added to the culture medium to inhibit bacterial contamination and growth.The suitable growth temperature for PichiaPink-2 yeast is 24-30℃, and temperatures exceeding 32℃ can be catastrophic for intracellular protein expression, potentially leading to cell death. The ADE2 gene encodes for the enzyme phosphoribosylaminoimidazole carboxylase, which catalyzes the sixth step in the synthesis of purine nucleotides. In Saccharomyces cerevisiae, Pichia pastoris, or other yeasts, mutations or deletions of this gene can lead to the accumulation of purine precursors in the vacuoles of yeast cells, resulting in a pink coloration of the cells. The Pink2 yeast strain is a nutrient-deficient ade2 type, where the ade2 gene is deleted, and part of the ade2 promoter is also deleted, preventing the strain from growing in media lacking adenine, and it grows slowly in rich media. After transforming this strain with an expression plasmid containing the ade2 gene, the strain can grow in media lacking adenine, and its color is restored from the pink of the ade2 gene deletion to white, indirectly indicating the successful transformation of those cloned strains with the plasmid. PichiaPink-2 is a pep4 gene knockout yeast strain. The pep4 gene encodes for protease A, an aspartic protease within the cell's vacuoles that can auto-activate. Since protease A plays an important role in the activity of other proteases within the cell's vacuoles, knockouts of the pep4 gene lead to a decrease in the activity of protease B, and the activity of carboxypeptidase Gama within the cell is also lost, which helps to protect the expression products from degradation and promotes an increase in expression levels.

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