Use of Modified BL21(DE3) Escherichia coli Cells for High-Level Expression of Recombinant Peanut Allergens Affected by Poor Codon Usage (1)
The cDNAs were subcloned into the expression vector pET-16b that uses the T7 RNA polymerase-responsive promoter to produce (His)10-tagged fusion proteins. This resulted in an ineffective expression of Ara h 1, Ara h 2, and Ara h 6 in conventional BL21(DE3) Escherichia coli cells. A complete compilation of codon usage of the sequences placed in the GenBank database can be found at http://www.kazusa.or.jp/codon/.
Only the peanut allergen Ara h 5 with a 396-bp cDNA-coding region and a predicted MW of 14 kDa was successfully expressed in these conventional E. coli cells. Greatly increased in the special BL21(DE3)CodonPlus-RIL cells as also shown by SDS– PAGE analysis and subsequent Coomassie blue staining. The IgE- binding capacity of the recombinant proteins was confirmed by Western blot analysis.
E. coli only uses three to four AGG/AGA codons per 1000 codons whereas the peanut allergens Ara h 1, Ara h 2, and Ara h 6 contain 54, 96, and 81 AGG/AGA codons, respectively, per 1000 codons. Until recently, three strategies were usually recom- mended to solve this problem): first, use a host con- taining a plasmid with the appropriate tRNA; second, synthesize the gene to replace rare codons with frequently used codons; or third, use a eukaryotic expression system. The fourth alternative is the use of E. coli hosts that carry extra copies of the argU tRNA gene.
Ara h 2 is partly enriched and aggregated in inclusion bodies in the cytoplasm of E. coli. The soluble fraction was purified by Ni–NTA metal-affinity chromatography under native conditions with imidazole using an FPLC apparatus.
The total yield of the eluted recombinant protein was estimated as 4.5 mg/liter culture. Pooling of the elution fractions from the Ni–NTA affinity chromatography of the soluble fraction as well as of the insoluble fraction yielded highly pure (.95% purity) recombinant (His)10- Ara h 2 with a total yield of approximately 28.5 mg/ liter culture. Concerning Ara h 2, containing seven cysteine resi- dues, the isolated inclusion body proteins usually contain a certain amount of interchain disulfide bonds which reduces the solubility of the proteins in the absence of reducing agents such as glutathione, cysteine, or 2-mercaptoethanol. Therefore, the dialysis was performed with 5 mM cysteine solution in order to have a favorable effect on renaturation with a minimal loss of purified protein of less than 10%.
