Recombinant E6 expressed in Escherichia coli is known to form recalcitrant inclusion bodies even when fused to the soluble GST protein. This study describes the modification of the HPV genotype-16 oncogenic protein E6 in order to obtain it in the soluble form. The modified protein (ΔE6) was expressed in E. coli BL21 as an N-terminal fusion with GST (GST-ΔE6). ΔE6 was constructed by deleting the nucleotide sequences coding for IHDIIL (31-36 a.a), one of the highly hydrophobic peptide stretches, using splicing by overextension polymerase chain reaction (SOE-PCR). The removal of IHDIIL residues rendered the GST-ΔE6 soluble and amenable for purification involving a two step process a preliminary glutathione-GST affinity chromatography followed by gel-filtration chromatography. Evaluation of purified protein fractions by HPLC suggests that GST-ΔE6 exists as a monomer. Further, the ΔE6 in GST-ΔE6 seemed to retain the binding ability to p53 as determined by the glutathione-GST capture ELISA. Purified GST-ΔE6 we reckon, might find use as an essential reagent in immunological assays, in sero-epidemiological studies, and also in studies to delineate the structure and function of HPV16 E6.
Keywords: GST-ΔE6; HPV16 E6; Oligomeric state; ProtScale and ProtParam analysis; SOE-PCR.
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