KM71 Chemically Competent Cell

KM71 Chemically Competent Cell

The KM71 strain is a Pichia pastoris strain specifically developed by Invitrogen for recombinant protein expression. KM71 is a histidine auxotrophic strain, with a mutation at the histidine dehydrogenase (His4) gene locus in its genome, making it unable to synthesize histidine. Some Pichia pastoris expression plasmids carrying the HIS selection marker (such as pPIC3.5K, pPIC9K, pAO815, etc.) can express the HIS4 gene and complement KM71, allowing for the selection of successfully integrated positive transformants through histidine-deficient medium. Other Pichia pastoris expression plasmids without the HIS selection marker (such as pPICZ A, B, C, pPICZα A, B, C, etc.) can also be used to select positive transformants through Zeocin or bleomycin hydrochloride. The phenotype of KM71 Pichia pastoris is MutS, Arg+, His-. His- indicates that KM71 is a histidine auxotrophic strain and cannot grow on histidine-deficient medium. Arg+ indicates that the parent strain of KM71 has a mutation in the arginine succinate lyase gene (arg4), and the introduction of the wild-type ARG4 gene at the AOX1 gene locus simultaneously disrupted the AOX1 gene, resulting in the MutS phenotype. MutS indicates that the AOX1 gene function of KM71 is disrupted (Pichia pastoris is a methanol-nutrient yeast that can use methanol as the sole carbon source. The first step of methanol metabolism is the oxidation of methanol to formaldehyde by alcohol oxidase. There are two genes in Pichia pastoris that encode alcohol oxidase: AOX1 and AOX2. In wild-type Pichia pastoris, most of the alcohol oxidase is the product of the AOX1 gene, and methanol can induce the expression of the AOX1 gene. The AOX1 protein product can account for more than 30% of the soluble proteins in the cell. Many Pichia pastoris expression plasmids use the AOX1 expression cassette for the expression of exogenous genes. If Pichia pastoris contains a functional AOX1 gene, it is called the Mut+ type; AOX2 is a homologous gene of AOX1 (97% homology). When the AOX1 gene is missing or loses its function, the AOX2 gene will take effect, generating a MutS mutant strain. However, the ability of AOX2 to oxidize methanol is much weaker than that of AOX1, resulting in a decrease in the cell's ability to metabolize methanol and slow growth in methanol medium. For a specific protein, it is difficult to predict whether choosing Mut+ or MutS as the host for protein expression is better. Although Mut+ type yeast transcribes more mRNA, excessive mRNA does not necessarily produce active proteins; although MutS type yeast grows slowly and transcribes less mRNA than Mut+, it may be more conducive to the formation of the correct protein conformation and the production of active proteins. KM71 chemically competent cells are specially prepared and can be stored at -80°C for 12 months. The transformation efficiency of linearized pPIC9K plasmid (9.3 kb, Amp^R in E. coli) is >10⁴ cfu/μg DNA.

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