BL21-CodonPlus(DE3)-RP E.coli菌株感受态细胞 BioVector NTCC质粒载体菌种细胞基因保藏中心

BL21-CodonPlus(DE3)-RP E.coli菌株感受态细胞 BioVector NTCC质粒载体菌种细胞基因保藏中心
BL21-CodonPlus(DE3)-RP strain Genotype E. coli B F– ompT hsdS(rB – mB – ) dcm+ Tetr gal λ(DE3) endA Hte [argU proL Camr
Efficient production of heterologous proteins in E. coli is frequently limited by the rarity of certain tRNAs that are abundant in the organisms from which the heterologous proteins are derived.
Forced high-level expression of heterologous proteins can deplete the pool of rare tRNAs and stall translation. BL21-CodonPlus strains are engineered to contain extra copies of genes that encode the tRNAs that most frequently limit translation of heterologous proteins in E. coli. Availability of tRNAs allows high-level expression of many heterologous recombinant genes in BL21-CodonPlus cells that are poorly expressed in conventional BL21 strains. BL21-CodonPlus-RIL and BL21-CodonPlus(DE3)-RIL cells contain extra copies of the argU, ileY, and leuW tRNA genes. These genes encode tRNAs that recognize the arginine codons AGA and AGG, the isoleucine codon AUA, and the leucine codon CUA, respectively (Table I). The CodonPlusRIL strains have available the tRNAs that most frequently restrict translation of heterologous proteins from organisms that have AT-rich genomes. BL21-CodonPlus-RP and BL21-CodonPlus(DE3)-RP cells contain extra copies of the argU and proL genes. These genes encode tRNAs that recognize the arginine codons AGA and AGG and the proline codon CCC, respectively. The CodonPlus-RP strains have available the tRNAs that most frequently restrict translation of heterologous proteins of organisms that have GC-rich genomes. The BL21-CodonPlus (DE3)-RIPL cells contain extra copies of the argU, ileY, and leuW as well as the proL tRNA genes. This strain rescues expression of heterologous proteins from organisms that have either AT- or GC-rich genomes. The BL21-CodonPlus(DE3) strains (Table II) are ideal for performing protein expression studies that utilize the T7 RNA polymerase promoter to direct high-level expression. The BL21-CodonPlus-RIL and BL21-CodonPlus-RP strains can be used for protein expression with vectors driven by non-T7 promoters. The methionine auxotrophic variants BL21-CodonPlus(DE3)-RIL-X and BL21-CodonPlus(DE3)-RP-X allow efficient labeling of recombinant proteins with selenomethionine or 35S-methionine. Both strains contain a transposon in the metA gene that renders the cells incapable of synthesizing methionine. Derived from E. coli B, the BL21-CodonPlus expression strains naturally lack the Lon protease, which can degrade recombinant proteins. In addition, these strains are engineered to be deficient for a second protease, the OmpT protein. BL21-CodonPlus competent cells also feature the Hte phenotype present in Agilent’s highest efficiency competent cell strain, XL10-Gold.2 The presence of the Hte phenotype increases the transformation efficiency of the cells. In addition, the gene that encodes endonuclease I (endA), an enzyme that rapidly degrades plasmid DNA isolated by most miniprep procedures, has been inactivated in these cells. These two features enable direct cloning of many protein expression constructs. Protein Expression Systems and Induction Methods ♦ The BL21-CodonPlus(DE3)-RIPL, BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP competent cells are all-purpose strains for high-level protein expression and easy induction in T7 expression systems. ♦ The strains of the BL21-CodonPlus series of competent cells provide varying levels of expression control with T7 promoterdriven vectors such as the pCAL vectors and the pET vectors (Table III). BL21-CodonPlus-RIL and BL21-CodonPlus-RP cells require infection with the CE6 bacteriophage for T7 promoterdriven expression. With expression induced by the CE6 bacteriophage, the BL21-CodonPlus-RIL and BL21-CodonPlus-RP cells provide the tightest control of protein expression. ♦ BL21-CodonPlus-RIL and BL21-CodonPlus-RP cells can be used for protein expression with vectors driven by non-T7 promoters. tRNA Expression Plasmids ♦ BL21-CodonPlus-RIL, BL21-CodonPlus(DE3)-RIL, and BL21- CodonPlus(DE3)-RIL-X cells contain a ColE1-compatible, pACYC-based plasmid containing extra copies of the argU, ileY, and leuW tRNA genes. ♦ BL21-CodonPlus-RP, BL21-CodonPlus(DE3)-RP, and BL21- CodonPlus(DE3)-RP-X cells contain a ColE1-compatible, pACYC-based plasmid containing extra copies of the argU and proL tRNA genes. ♦ BL21-CodonPlus(DE3)-RIPL cells contain a ColE1-compatible, pACYC-based plasmid containing extra copies of the argU and proL tRNA genes and a ColE1- and pACYC-compatible pSC101- based plasmid containing extra copies of the argU, ileY, and leuW tRNA genes. Protein Labeling Strains ♦ BL21-CodonPlus(DE3)-RIL-X and BL21-CodonPlus(DE3)-RP-X cells are methionine auxotrophic variants of the BL21- CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP strains, respectively, and are designed to allow efficient labeling of recombinant proteins with selenomethionine or 35S-methionine.
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