High Yield Expression and Modified Purification of Novel Recombinant Truncated Protein FimH.MrpH against Urinary Tract Infections by Escherichia coli and Proteus mirabilis KC06-KC09
Dr. Saeid Bouzari,
Professor, Department of Molecular Biology, Pasteur Institute of Iran, Pasteur Ave, Tehran-13164, Iran.
Introduction: Urinary Tract Infections (UTIs) rank third among the list of most common bacterial infections. FimH and MrpH are the most important antigens of Escherichia coli and Proteus mirabilis isolates, respectively.
Aim: To design a novel truncated fusion protein of immunogenic virulence factors and to assess expression efficiency of fusion protein made.
Materials and Methods: N-terminal domains of MrpH (first 405 nucleotides) and FimH (first 495 nucleotides) that have functional and antigenic properties were selected to make a fusion protein. In silico and bioinformatics studies were designed to determin physico-chemical parameters. The Fusion gene was amplified by overlap PCR. After sequencing, it was inserted and expressed into pET28a vector. The expressed protein was purified by Ni-NTA column according to a modified protocol.
Results: According to in silico studies, fusion tMrpH.tFimH was predicted as high quality model. About 900 bp long fragment was amplified by overlap PCR. Enzyme digestion and sequencing confirmed the cloning of the fusion gene in pET28a vector. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed approximately 30 kDa band and Western blot confirmed the presence of fusion protein. A modified purification method has been tested which resulted in higher yield with high level of purity.
Conclusion: In order to have effective vaccine against UTI, we must study a wide variety of strategies. This study provides the new protocol for efficient purification of tMrpH.tFimH protein in which the quantity and purification of protein is high. This recombinant protein could be introduced as vaccine candidate in the future study.