( formed a series of experiments to demon...)New BL21-CodonPlus Cells Correct Codon Bias in GC-Rich Genomes

formed a series of experiments to demonstrate the effectiveness of the BL21-CodonPlus strains in rescuing expression of genes that are affected by rare codon usage. We transformed BL21-CodonPlus(DE3)-RIL cells and BL21-Gold(DE3) cells with plasmids encoding the following genes regulated by the T7 RNA polymerase-responsive promoter (Figure 1): human cardiac troponin-T (hcTnT-wt) ⁵, yeast Hsp 104, and four genes of archael origin. For each recombinant gene, protein synthesis greatly increased in the BL21-CodonPlus(DE3)-RIL cells, as compared to BL21-Gold(DE3) cells. Expression of the control genes, which do not encode rare codons (l-phosphastase and c-Jun N-terminal kinase (JNK), was equivalent in BL21-CodonPlus(DE3)-RIL and BL21-Gold(DE3) cells. Moreover, extra copies of the tRNA genes did not cause any obvious deleterious effects to the host cells. Accordingly, BL21-CodonPlus cells can also be used as hosts for expressing genes that are expressed well in conventional E. coli strains.

Expression of Genes from GC-Rich Organisms

To confirm the function of the argU and proL tRNAs in BL21-CodonPlus(DE3)-RP cells, we performed two test transformations (Figure 2). BL21-CodonPlus(DE3)-RP and BL21-Gold(DE3) cells were transformed with plasmids encoding the hcTnT-wt (argU dependent) or CBP-3xP-Cre (proL dependent) genes. The BL21-CodonPlus(DE3)-RP cells, but not the parental BL21-Gold(DE3) cells, restored expression of both genes. These data demonstrate the suitability of BL21-CodonPlus(DE3)-RP cells for high-level expression of heterologous genes restricted by the presence of AGG/AGA and CCC codons in conventional BL21 hosts.

Fig.2

Conclusion
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