( series (Novagen)that uses the T7 RN...)High-Level Expression of Peanut Allergens Affected by Rare Codon Usage

series (Novagen) that uses the T7 RNA polymerase responsive promoter to produce (His)₁₀ -tagged fusion proteins. Two E. coli strains were transformed with this plasmid and selected on LB broth supplemented with ampicillin: BL21-Gold(DE3) cells and the recently generated BL21-CodonPlus (DE3)-RIL cells ⁶ carrying extra copies of the argU, ileY, and leuW tRNA genes. Recombinant Ara h 2 expression was induced by adding 1 mM IPTG when the cells reached an optical densitiy of 0.6 to 0.8 at 600 nm. Incubation was continued at three different temperatures (22C, 30C, 37C) for an additional 2, 4, and 20 hours.

The production of Ara h 2 was enhanced significantly with the new BL21-Codon Plus (DE3)-RIL cells (Figure 1C), compared to the pQE/M15 system (Figure 1A) and to the conventional BL21-Gold(DE3) cell expression system (Figure 1B). The highest amount of recombinant protein was achieved by selecting an overnight incubation at 22C after IPTG induction.

The final yield of soluble recombinant Ara h 2 was estimated as 5 mg/L of culture, which is 100 times greater when compared to the 50 g achieved by the pQE/M15 expression system.

Conclusions

It is possible to make large quantities of high-quality heterologous proteins that are otherwise difficult to express in conventional E.coli hosts. When low-expression yields or low-quality recombinant protein is due to rare codon usage, BL21-CodonPlus (DE3)-RIL cells are the hosts of choice. We used these special cells and greatly increased the expression yield of the major peanut allergen Ara h 2, which contains 12% of the rare AGG/AGA codons in its message.

Methods

A 10-ml culture of each of the expression experiments was induced by adding 1 mM IPTG, then incubation commenced at three different temperatures (22C, 30C,
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