SMD1168H Chemically Competent Cell

For the first use of Carrier DNA，boil the tube containing Carrier DNA in boiling water for 5 minutes，then place it on ice immediately. After use，store it at -20℃ for later standby. Thaw Carrier DNA on ice before next use. Re-denature Carrier DNA after 3 to 5 repeated freeze-thaw cycles. The pPICZ A plasmid needs to be integrated into the Pichia pastoris genome. It must be linearized by single restriction enzyme digestion before transformation，and SalI is commonly used for linearization. A concentration of 100 μg/mL Zeocin/Bleomycin Hydrochloride can be adopted for screening single-copy insertion of the Zeocin resistance gene into the Pichia pastoris genome. Multi-copy integration can enhance the resistance level of Pichia pastoris to Zeocin/Bleomycin Hydrochloride. The concentration can be increased according to experimental needs to screen strains with high-copy protein expression cassettes. Pichia pastoris SMD1168H is temperature-sensitive with an optimal growth temperature of 27℃-30℃; temperatures above 31℃ will affect the growth of yeast. KM71 is a Pichia pastoris strain specially used for recombinant protein expression. SMD1168H can express the HIS4 gene and cannot utilize auxotrophic medium to screen recombinant positive colonies; Pichia pastoris expression plasmids carrying Zeocin resistance genes such as pPICZ A，B，C and pPICZα A，B，C can be screened with 100 ug/mL Zeocin or bleomycin hydrochloride. The phenotype of SMD1168H is Mut+，Pep4-; Pep4- indicates proteinase A deficiency，which helps the stable expression of target proteins，and Mut+ means SMD1168H has normal functional AOX1 gene. There are two genes encoding alcohol oxidase in Pichia pastoris: AOX1 and AOX2. Most alcohol oxidase in wild-type Pichia pastoris is the product of the AOX1 gene，methanol can induce the expression of AOX1，and the AOX1 protein product can account for more than 30% of the soluble protein in cells. Many Pichia pastoris expression plasmids utilize the AOX1 expression cassette for exogenous gene expression，and strains with functional AOX1 gene are defined as Mut+ type. AOX2 is a homologous gene of AOX1 with 97% homology; when the AOX1 gene is deleted or loses function，the AOX2 gene will function and generate MutS mutant strains. However，the ability of AOX2 to oxidize methanol is much weaker than that of AOX1，resulting in reduced methanol metabolism capacity and slow growth in methanol medium. For different proteins，it is difficult to predict whether Mut+ or MutS is more suitable as the host for protein expression. Although Mut+ yeast transcribes more mRNA，excessive mRNA does not necessarily produce biologically active proteins; MutS yeast grows slowly and transcribes less mRNA than Mut+，but it may be more conducive to the correct conformation of proteins and produce biologically active proteins. The transformation efficiency of SMD1168H chemically competent cells detected with linearized pPICZ A plasmid (3. 3kb，ZeocinR in E. coli/Pichia pastoris) is >104. Genotype: pep4; Phenotype: Mut+，Pep4-