Shedding Light onH. pyloriDetection: A Fusion Protein Approach Unveiled through LIPS Method

Abstract

ABSTRACTThe Luciferase Immunoprecipitation Systems (LIPS) method serves as a highly sensitive approach for quantitatively detecting antibodies to antigens, offering potential in identifying viral and bacterial infections. However, the substantial size of the luciferase-antigen fusion protein presents challenges in both production and folding. An alternative strategy employing epitopes rather than full length antigenic protein may circumvent issues associated with recombinant expression.Helicobacter pylori, a gram-negative bacterium, poses a risk of gastric cancer if untreated over time. This study focuses on the recombinant production of a fusion protein comprising in silico designed antigenic epitopes from theH. pyloriurease protein and luciferase, aiming to reduce the fusion protein’s size and thus augment its expression in theE. colisystem. By employing bioinformatic analysis, sequences encoding the antigenic regions were pinpointed and subsequently amplified via PCR. A luciferase-linker-epitope construct was devised and constructed accordingly. TheE. coliBl21 (DE3) strain was utilized to express the recombinant chimeric protein, which was subsequently purified to achieve a state of homogeneity. The molecular weight of the fusion protein was estimated to be 75 kilodalton. Verification of the chimeric protein’s proper folding and functionality was confirmed, as evidenced by a bioluminescence assay yielding an emission of 13.7 × 106(RLU/s). Furthermore, western blot analysis authenticated the fusion protein’s capability to bind specifically toH. pyloriantibodies. These findings underscore the potential of the resultant protein as a promising candidate forH. pyloridetection while also streamlining the recombinant production of LIPS fusion proteins.Key PointsEpitope-driven protein design boostsE. coliexpression for LIPS advancement.ImprovedH. pyloridetection aids early gastric cancer identification.