SMD1168H Chemically Competent Cell

SMD1168H Chemically Competent Cell

The SMD1168H strain is a Pichia pastoris strain developed by Invitrogen specifically for the expression of recombinant proteins. SMD1168H can express the HIS4 gene and positive colonies with successful recombination can be screened using nutritionally deficient media; Pichia pastoris expression plasmids such as pPICZ A, B, C, pPICZα A, B, C, which contain the Zeocin resistance gene, can be screened using 100 µg/ml of Zeocin or hygromycin B. The phenotype of SMD1168H Pichia pastoris is Mut, Pep4–, where Pep4– indicates a defect in proteinase A, encoded by the Pep4 gene, which is an aspartic protease that can auto-activate in the vacuole. Since proteinase A can promote the activation of other proteases, the activity of protease B is also reduced in the pep4-deficient strains, helping in the stable expression of the target protein. Mut indicates that SMD1168H contains a functional AOX1 gene (Pichia pastoris is a methanol-utilizing yeast that can use methanol as the sole carbon source. The first step of methanol metabolism is: alcohol oxidase uses oxygen molecules to oxidize methanol to formaldehyde, with two genes in Pichia pastoris coding for alcohol oxidases: AOX1 and AOX2. Most alcohol oxidase in wild-type Pichia pastoris is the product of the AOX1 gene, and methanol can induce the expression of the AOX1 gene, with the product of the AOX1 protein potentially constituting over 30% of the soluble protein in the cells. Many Pichia pastoris expression plasmids utilize the AOX1 expression frame for foreign gene expression. If Pichia pastoris has a functional AOX1 gene, it is referred to as Mut type; AOX2 is a homologous gene of AOX1 (97% homology) and will take effect when the AOX1 gene is deleted or becomes nonfunctional, producing a MutS phenotype mutant. However, AOX2 has a weaker ability to oxidize methanol than AOX1, leading to a decreased capacity for methane metabolism and slower growth in methanol media. It is difficult to predict whether Mut or MutS would be a better host for protein expression for a specific protein. Although Mut type yeast yields more transcribed mRNA, it does not necessarily produce functional proteins. While MutS type yeast grows slowly and transcribes less mRNA than Mut, it may be more favorable for the correct conformation of proteins, leading to the production of active proteins.) SMD1168H competent cells are created through a special process, and the linearized pPICZ A plasmid (3.3 kb, Zeocin^R in E. coli/Pichia pastoris) shows a transformation efficiency greater than 10⁴ cfu/µg DNA.

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