Full-size recombinant ORF1p-L1 and RT domain of ORF2p-L1: Protein expression, purification and characterization

Long interspersed nuclear elements-1 (LINEs-1) are known to be active human retrotransposons containing two protein-coding genes, ORF1 and ORF2, whose expression results in the production of two proteins-ORF1p-L1 and ORF2p-L1, respectively. Activation of LINE-1 may occur during the early stages of lung, prostate, esophageal, colon or breast cancer progression, often without obvious pathological symptoms or any overt signs of disease. In this study, we developed a method for preparing the LINE-1 proteins ORF1p-L1 and the reverse transcriptase (RT) domain of ORF2p-L1 and demonstrated their potential application as antigens in gastric cancer diagnostics. Both antigens were expressed as insoluble inclusion bodies in a bacterial expression system (E. coli BL21 (DE3) pLysS) using laboratory-scale fermentation. The preparation protocol involved solubilizing the inclusion bodies with a chaotropic agent, followed by multiple protein purification steps and subsequent renaturation of the proteins by dialysis under acidic conditions. At decreasing concentrations of the chaotropic agent, ORF1p-L1 and the RT domain of ORF2p-L1 were found to weakly bind to cation-exchange resins or to aggregate, whereas irreversible protein binding occurred under acidic conditions. The biological activity of the refolded ORF1p-L1 was confirmed by assessing its hybridization activity, while the reverse transcriptase activity of the RT domain of ORF2p-L1 was also successfully confirmed. Both refolded proteins reveal antigen-antibody response against antibodies in serum samples of patients with gastric cancer.