Shedding light on H. pylori detection: A fusion protein approach unveiled through LIPS method

Abstract

The Luciferase Immunoprecipitation Systems (LIPS) method is a highly sensitive approach for quantitatively detecting antibodies, offering potential in identifying viral and bacterial infections. However, the substantial size of the luciferase-antigen fusion protein presents challenges in production and folding. Using epitopes rather than the full-length antigenic protein may circumvent issues with recombinant expression. Helicobacter pylori, a gram-negative bacterium, poses a risk of gastric cancer if untreated. This study focuses on producing a fusion protein comprising in silico-designed antigenic epitopes from the H. pylori urease protein and luciferase, aiming to reduce the fusion protein's size and augment its expression in the E. coli system. Bioinformatic analysis identified sequences encoding antigenic regions, which were amplified via PCR. A luciferase-linker-epitope construct was then devised and expressed in the E. coli Bl21 (DE3) strain. The recombinant protein was primarily purified to homogeneity, yielding a major band at 75 kilodaltons. Verification of the protein's proper folding and functionality was confirmed through a bioluminescence assay with an emission of 13.7 × 10^6 RLU/s. Western blot analysis authenticated the fusion protein's specific binding to H. pylori antibodies. These findings underscore the potential of the protein as a promising candidate for H. pylori detection and streamline LIPS fusion protein production.