KM71 Chemically Competent Cell

1.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. Store the remaining reagent at -20℃ for future use after application. Thaw Carrier DNA on ice before each subsequent use. Re-denature Carrier DNA after 3 to 5 repeated freeze-thaw cycles; 2. The pPIC9K plasmid needs to integrate into the Pichia pastoris genome. It must be linearized via single restriction enzyme digestion before transformation，and SalI is the most commonly used enzyme for linearization; 3. A concentration of 100 μg/mL Zeocin (Bleomycin Hydrochloride) can be used for screening single-copy insertion of the Zeocin resistance gene into the Pichia pastoris genome. Multi-copy integration can enhance the resistance of Pichia pastoris to Zeocin/Bleomycin Hydrochloride. Accordingly，the concentration of Zeocin/Bleomycin Hydrochloride can be increased as required by experiments to screen strains with high-copy protein expression cassettes; 4. Pichia pastoris KM71 is temperature-sensitive. Its optimal growth temperature is 27℃-30℃，and temperatures higher than 31℃ will adversely affect the growth of yeast. KM71 is a Pichia pastoris strain specially used for recombinant protein expression. As a histidine auxotrophic strain，KM71 has a mutation at the genomic histidine dehydrogenase (His4) locus and cannot synthesize histidine by itself; some Pichia pastoris expression plasmids carrying the HIS selection marker such as pPIC3. 5K，pPIC9K and pAO815 can express the HIS4 gene to complement KM71，allowing positive integrants 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 to screen positive transformants with Zeocin or bleomycin hydrochloride. The phenotype of KM71 is Mut?，Arg?. The parent strain of KM71 has a mutation in the argininosuccinate lyase gene (arg4)，and the wild-type ARG4 gene is inserted into the AOX1 locus，endowing KM71 with the Arg? phenotype; meanwhile，the insertion of ARG4 disrupts the AOX1 gene，resulting in the Mut? phenotype. 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 can account for more than 30% of the total 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; when AOX1 is deleted or non-functional，AOX2 takes effect and forms Mut? mutant strains. However，the methanol oxidation capacity of AOX2 is much weaker than that of AOX1，leading to reduced methanol metabolism ability and slow growth in methanol medium. It is difficult to predict whether Mut? or Mut? is more suitable as the host strain for the expression of different proteins. Although Mut? yeast produces more transcribed mRNA，excessive mRNA does not necessarily yield biologically active proteins; while Mut? yeast grows slowly and produces less mRNA than Mut?，it may be more conducive to the correct conformational folding of proteins and the production of biologically active proteins. The transformation efficiency of KM71 chemically competent cells tested with linearized pPIC9K plasmid (9. 3 kb，Amp? in E. coli) is higher than 10? cfu/μg DNA. Genotype: his4，aox1::ARG4，arg4; Phenotype: Mut?，Arg?，His?.