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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