Construction, production and identification of rE7m and rlipo-E7m.

<p>(<b>a</b>) The alignment of the amino acid sequence of wild-type HPV16 E7 (Accession number: NP_041326) and the inactive E7 (E7m). Every mutant site of E7m is highlighted. The DNA sequence of the E7m gene was derived using codon usage of <i>E. coli</i> and fully synthesized using the assembly PCR method. The PCR product was cloned into the pET22b vector for rE7m expression and into the modified pET22b vector with a lipid signal peptide for rlipo-E7m expression. The recombinant proteins contained an additional HHHHHH sequence (HisTag) at the C-terminus and were expressed under the control of the T7 promoter. (<b>b</b>) The rlipo-E7m and rE7m protein purification process was monitored using 15% reducing SDS-PAGE followed stained by Coomassie Blue staining and immunoblotting using anti-HisTag antibodies. The recombinant rlipo-E7m was expressed in <i>E. coli</i> strains C43 (DE3). Lane 1, rE7m expression after IPTG induction; lane 2, protein expression in the absence of IPTG induction; lane 3, extracted fraction of rE7m; lane 4, purified recombinant rE7m. Lanes 5–8 show immunoblotting to monitor the rE7m purification process, and the samples in these lanes are the same as those in lanes 1–4, respectively. Lane 9, rlipo-E7m expression after IPTG induction; lane 10, protein expression in the absence of IPTG induction; lane 11, purified rlipo-E7m. Lanes 12–14 show immunoblotting to monitor the rlipo-E7m purification process, and the samples in these lanes are the same as those in lanes 9–11, respectively. The non-lipidated form of rE7m was expressed in <i>E. coli</i> BL21 (DE3) star strain. The arrows indicate the electrophoretic positions of rE7m or rlipo-E7m in the gels or blots. (<b>c</b>) Intact rlipo-E7m was analyzed by LC/MS. LC/MS analysis revealed the presence of five major post-translational modifications of rlipo-E7m. Molecular masses (in daltons) were determined using a maximum entropy algorithm. The five major peaks displayed masses of 15384.5, 15400, 15412.5, 15426 and 15439. (<b>d</b>) N-terminal rlipo-E7m fragments were obtained and identified after digestion of rlipo-E7m with trypsin. The digested sample was analyzed on a Waters® MALDI micro MX™ mass spectrometer. The MALDI-TOF MS spectra revealed the existence of five peaks with m/z values of 1452, 1466, and 1480, 1492 and 1506.