( Produce large quantities of di...)High-Level Expression of Peanut Allergens Affected by Rare Codon Usage

Produce large quantities of difficult-to-express proteins

Tamara Kleber-Janke Wolf-Meinhard Becker
Biochemical & Molecular Allergology, Research Center Borstel, Germany

By using Stratagenes BL21-CodonPlus(DE3)-RIL cells * with extra copies of E. coli, argU, ileY, and leuW tRNA genes, it is possible to attain high-level expression of difficult proteins affected by poor codon usage: IPTG-induced expression of several recombinant peanut allergen genes, such as Ara h 2, were greatly increased in these special cells compared to the expression yield achieved by conventional E. coli cells like M15 or BL21-Gold(DE3) cells.

E. coli remains a popular host for the expression of heterologous proteins. Although the E. coli cell has a tremendous capacity to produce large quantities of protein, there are limits when the composition of the mRNA or protein is not typical. Within E. coli, a clear bias exists among the 61 amino acid codons found within the population of mRNA molecules, and the level of cognate tRNA appears directly proportional to the frequency of codon usage.¹

A subset of codons, namely AGG/AGA, AUA, CUA, CGA, and CCC, are the least used codons in E. coli that are encoded by rare tRNAs. Recent studies suggest that an excess of any of these codons creates problems during translation, leading to a reduction in quantity of the protein synthesized.² Errors are detectable as frameshifts and decreased levels of expression. More subtle changes, such as mistranslation, may also be occurring.

Until recently, three alternatives were usually recommended to solve these problems: Use a host containing a plasmid with the appropriate tRNA,³ synthesize the gene to replace rare codons with frequently used codons, and use aeukaryotic expression system.

Comparing Different Expression Vectors and E. coli Strains