SMD1168 Chemically Competent Cell

For the first use of Carrier DNA，boil the tube containing Carrier DNA in boiling water for 5 min，then place it on ice immediately. Store it at -20℃ for standby after use. Thaw Carrier DNA on ice before subsequent use. Re-denature Carrier DNA after 3-5 repeated freeze-thaw cycles. The pPIC9K plasmid needs to be integrated into the Pichia pastoris genome. It must be linearized by single enzyme digestion before transformation，and SalI is routinely used for linearization. Single-copy insertion of the Zeocin resistance gene into the Pichia pastoris genome can be screened at 100 μg/mL Zeocin/Bleomycin Hydrochloride. Multi-copy integration can improve the resistance of Pichia pastoris to Zeocin/Bleomycin Hydrochloride. The concentration can be increased according to experimental requirements to screen strains with high-copy protein expression cassettes. Pichia pastoris KM71 is temperature-sensitive. The optimal growth temperature is 27℃-30℃; temperatures above 31℃ will affect the growth of yeast. SMD1168 is a Pichia pastoris strain specially applied for recombinant protein expression. As a histidine auxotrophic strain，SMD1168 has a mutation at the genomic histidine dehydrogenase (His4) locus and cannot synthesize histidine on its own; certain Pichia pastoris expression plasmids carrying the HIS selection marker such as pPIC3. 5K，pPIC9K and pAO815 can express the HIS4 gene to complement SMD1168，allowing positive integrated transformants to be screened on histidine-deficient medium，while other Pichia pastoris expression plasmids without the HIS selection marker including pPICZ A，B，C and pPICZα A，B，C can also be used for screening positive transformants with Zeocin or bleomycin hydrochloride. The phenotype of SMD1168 is Mut?，Pep4?，His?; His? means SMD1168 is a histidine auxotrophic strain that cannot grow on histidine-deficient medium，Pep4? indicates proteinase A deficiency，which is conducive to the stable expression of target proteins，and Mut? means SMD1168 harbors a 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，whose expression can be strongly induced by methanol，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 are defined as Mut? type. AOX2 is a homologous gene of AOX1 with 97% sequence homology; it will function when AOX1 is deleted or loses its function，generating Mut? mutant strains. However，the methanol oxidation capacity of AOX2 is much weaker than that of AOX1，resulting in decreased methanol metabolism ability and slow growth in methanol medium. It is difficult to predict whether Mut? or Mut? is more suitable as the host for protein expression of different proteins. Although Mut? yeast transcribes more mRNA，excessive mRNA does not necessarily produce biologically active proteins; while Mut? yeast grows slowly and generates less mRNA than Mut?，it may be more favorable for proteins to form correct conformations and produce biologically active proteins. The transformation efficiency of SMD1168 chemically competent cells verified with linearized pPIC9K plasmid (9. 3 kb，Amp? in E. coli) is over 10? cfu/μg DNA. Genotype: his4，pep4; Phenotype: Mut?，Pep4?，His?.