( justed hcTnT-mut in conventional ...)Codon Bias-Adjusted BL21 Derivatives for Protein Expression

justed hcTnT-mut in conventional BL21-Gold(DE3) cells. Hence, using BL21-CodonPlus(DE3)-RIL strains are as efficient as employing the conventional and time-consuming method of synonymous replacement of rare codons to achieve high-level expression of codon-usage restricted recombinant genes.

Fig.2

The simultaneous expression of several rare tRNA genes in BL21-CodonPlus(DE3)-RIL not only provides a generic host for expression of recombinant genes affected by a range of rare codons but is also necessary for some recombinant genes to achieve a high level of expression. In Figure 2, using an archaeal gene as an example, efficient production of this recombinant gene in E. coli hosts requires the simultaneous presence of functional extra copies of both argU and ileY tRNA genes. Variants of the BL21-CodonPlus(DE3)-RIL strains lacking either the argU or ileY gene, while retaining the other tRNA genes, failed to produce the target gene at the same level as observed in the BL21-CodonPlus(DE3)-RIL strain. The functional expression argU and ileY genes in each of thevariant strains were confirmed using expression constructs, which are dependent only on either argU function (hcTnT) or ileY function (3xi) for efficient expression. The artificial construct 3xi is derived from a well-expressed recombinant gene by insertion of three AUA codons (ile) at the N-terminus.

The host cells did not suffer any obvious deleterious effects from the extra copies of tRNA genes. Growth rates and transformation efficiencies were comparable to the parental BL21-Gold cells. When comparing BL21-CodonPlus(DE3)-RIL cells with BL21-Gold(DE3) cells, production of recombinant genes like
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