( Several results from expression exp...)High-Level Expression of Peanut Allergens Affected by Rare Codon Usage

Several results from expression experiments suggest that arginine codons AGG and AGA can have pronounced effects on the translation efficiency of cloned genes in E. coli.² These codons are the least frequently used in E. coli, and the tRNAs that recognize them are among the least abundant. Given this situation, we predicted translational problems with the mRNA of some of the recently identified peanut allergens ⁴ that contain an excess of rare, low-abundance tRNA codons. Hence, to achieve high-level expression of the peanut allergen Ara h 2, we tested different expression systems and compared the final yield of recombinant protein.

Ara h 2 cDNA was first subcloned into an expression vector of the pQE series (Qiagen) to produce (His)₆-tagged fusion proteins in E. coli M15 cells. In Figure 1A, expression of Ara h 2 protein was examined following 0, 2, and 4 hours as well as an overnight incubation once induction was completed. The Ara h 2 protein was visualized by immunoblot experiments (Figure 1A). No additional protein band of the appropriate molecular weight was detected after IPTG induction, demonstrating that Ara h 2 is only marginally expressed in these conventional E. coli cells.

Ara h 2 Codons

Examination of the codons used in this sequence indicated that Ara h 2 contains 12% of the least used codon AGG/AGA in E. coli and, additionally, 2% of other rare codons read by minor tRNAs within a message encoding a protein made up of 157 amino acids. This might explain the inefficient translation (also found by other authors) when truncated translation products from peanut allergen Ara h 1 are present.⁵

High-Level Expression in BL21-CodonPlus Cells

The Ara h 2 cDNA was subcloned into an expression vector of the pET
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