PichiaPink-4 Chemically Competent Cell

PichiaPink-4 Chemically Competent Cell

PichiaPink-4 is a Pink strain of Pichia pastoris, which belongs to eukaryotic cells. General antibiotics targeting prokaryotes, such as kanamycin and ampicillin, are ineffective against yeast; therefore, in order to prevent contamination of yeast culture strains by prokaryotes such as Escherichia coli, antibiotics like ampicillin and kanamycin are often added to the culture medium to inhibit bacterial contamination and growth. The suitable growth temperature for PichiaPink-4 yeast is 24-30℃. Temperatures exceeding 32℃ can have a disastrous impact on protein expression within the cells, potentially leading to cell death. The ADE2 gene encodes phosphoribosylaminoimidazole carboxylase, which catalyzes the sixth reaction in purine nucleotide synthesis. In Saccharomyces cerevisiae, Pichia pastoris, or other yeasts, mutations or deletions in this gene can lead to the accumulation of purine precursors within the vacuoles of yeast cells, resulting in a pink appearance. The Pink4 yeast strain is an ade2 nutritional deficiency type, with the ade2 gene deleted and part of the ade2 promoter also missing. As a result, the strain is unable to grow on adenine-deficient media and grows slowly on rich media. After introducing an expression plasmid containing the ade2 gene into this strain, it enables the strain to grow in adenine-deficient media, and the strain's color changes from pink, due to the deletion of the ade2 gene, back to white. This indicates the successful transformation of the plasmid into the cloned strains. PichiaPink-4 is a pep4 gene knockout yeast strain. The pep4 gene encodes protease A, which is an aspartic protease active in the vacuoles of the cell and can auto-activate. Because protease A plays a significant role in relation to other proteases in the cell's vacuoles, strains that have undergone the pep4 gene knockout show decreased activity of protease B, and the activity of carboxypeptidase Gama is also lost, thus protecting the expressed products from degradation and promoting an increase in expression levels. PichiaPink-4 is also a prb1 gene knockout yeast strain. The prb1 gene encodes protease B, which is a serine protease in the vacuoles of the cell and belongs to the subtilisin protease family. The absence of protease B in the cells can protect expressed products from degradation and promote an increase in expression levels. Both proteases A and B in PichiaPink-4 have been knocked out; thus, it is the strain with the lowest protease activity among the Pink series strains, effectively inhibiting and preventing protein degradation.

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