GS115 Chemically Competent Cell

GS115 Chemically Competent Cell

The GS115 strain is a Pichia pastoris strain developed by Invitrogen specifically for recombinant protein expression. GS115 is a histidine-deficient strain, with mutations at the histidine dehydrogenase (His4) gene locus in its genome, which means it cannot synthesize histidine on its own; some Pichia pastoris expression plasmids that carry a HIS selection marker (such as pPIC3.5K/pPIC9K/pAO815, etc.) can express the HIS4 gene, complementing GS115, and positive transformants can be screened using a histidine-deficient medium; other Pichia pastoris expression plasmids without a HIS selection marker (such as pPICZ A, B, C, pPICZα A, B, C, etc.) can also select for positive transformants using Zeocin or hygromycin B. The phenotype of GS115 Pichia pastoris is His–, Mut, where His– indicates that GS115 is a histidine-deficient strain that cannot grow in histidine-deficient medium; Mut indicates that GS115 has a functional AOX1 gene (Pichia pastoris is a methanol-utilizing yeast that can use methanol as its sole carbon source. The first step of methanol metabolism is: alcohol oxidase uses molecular oxygen to oxidize methanol to formaldehyde; there are two genes in Pichia pastoris that code for alcohol oxidase: AOX1 and AOX2. Most of the alcohol oxidase in wild-type Pichia pastoris is derived from the AOX1 gene product. Methanol can induce the expression of the AOX1 gene, and the AOX1 protein product can constitute more than 30% of the soluble proteins in cells. Many Pichia pastoris expression plasmids utilize the AOX1 expression frame for the expression of foreign genes. If Pichia pastoris contains a functional AOX1 gene, it is termed Mut type; AOX2 is a homologous gene to AOX1 (97% homology), and it will take effect when the AOX1 gene is deleted or loses its function, producing a mutant strain with a MutS phenotype. However, the ability of the AOX2 gene to oxidize methanol is significantly weaker than that of AOX1, resulting in a decreased capability to metabolize methanol and slow growth in methanol medium. It is difficult to predict whether to choose Mut or MutS as hosts for protein expression for specific proteins; while the Mut type yeast transcribes more mRNA, an excess of mRNA does not necessarily yield active proteins. Although the MutS type yeast grows slowly and produces less mRNA than Mut, it may be more favorable for the proteins to form the correct conformation, yielding active proteins.) The GS115 chemically competent cells are prepared using a special process, can be stored at -80°C for 12 months, and the linearized pPIC9K plasmid (9.3 kb, Amp^R in E. coli) has a transformation efficiency greater than 10⁴ cfu/μg DNA.

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