( sing site-directed mutagenesis or by su...)Codon Bias-Adjusted BL21 Derivatives for Protein Expression

Now, Stratagene has generated BL21-CodonPlus-RIL chemically competent cells that carry extra copies of the argU, ileY, and leuW tRNA genes. The tRNAs encoded by these genes recognize the AGA/AGG, AUA, and CUA codons, respectively. To demonstrate their ability to facilitate the expression of heterologous recombinant genes that are only marginally expressed in conventional BL21 strains, BL21-CodonPlus(DE3)-RIL cells were transformed with vectors expressing genes fromhuman, yeast, and archaeal origins using the T7 RNA polymerase responsive promoter. IPTG-induced expression of all recombinant genes greatly increased in the BL21-CodonPlus(DE3)-RIL cells, compared to their expression in BL21-Gold(DE3) (Figure 1).

Fig.1

Expression of Difficult Proteins

To demonstrate the efficiency of BL21-CodonPlus-RIL cells to facilitate protein production in genes affected by poor codon usage, we compared the expression of a codon-adjusted form of the human cardiac troponin-T gene in conventional BL21-GoldDE3 cells (Figure 1,hcTnT-mut) with expression of the wild-type (hcTnT-wt) form in BL21-CodonPlus(DE3)-RIL cells. The expression of hcTnT in E. coli was previously shown to be solely restricted by the presence of two rare arginine codon pairs.⁴ This was demonstrated by synonymous replacement of the rare arginine codon pairs with frequently used arginine codons (hcTnT-mut), which results in efficient production of hcTnT-mut in E. coli. In Figure 1, BL21-CodonPlus(DE3)-RIL cells express hcTnT-wt at the same level observed for the codon-ad
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