Cloning and expression of recombinant outer membrane protein A of Orientia tsutsugamushi using Escherichia coli expression systems

OmpA of Orientia tsutsugamushi (OtOmpA) plays an important role during multiple stages of scrub typhus pathogenesis. The bacterial expression system which involves cloning, is commonly used for the production of recombinant proteins. The cloning technique requires appropriate optimization and selection of an efficient expression vector. Therefore, this study aims to identify a suitable expression vector for the production of recombinant OtOmpA (rOtOmpA). Three different expression vectors with different properties were selected: pET-14b(+), pET-20b(+) and pET-28a(+). The DNA sequence of OtOmpA was cloned into the vectors and transformed into the TOP10 cloning host, respectively. Positive colonies carrying the recombinant plasmid were identified by using polymerase chain reaction and agarose gel electrophoresis. DNA sequencing was performed to verify the sequence of OtOmpA. Subsequently, each recombinant plasmid was transformed into the BL21(DE3)plySS expression host respectively. The rOtOmpA protein was expressed, extracted and analyzed by SDS-PAGE and Western blot. Analysis with anti-His tag antibodies showed only pET 20b(+) expresses rOtOmpA, which was confirmed by one-dimensional capillary liquid chromatography with tandem mass spectrometry (1D-LCMS/MS). Only pET-20b(+) possessed a pelB leader coding sequence incorporated in the upper region, closer to the ribosome binding site. The presence of the pelB leader may support the production of rOtOmpA, making pET-20b(+) a compatible expression vector. The production of rOtOmpA is crucial for further analysis of its role in the pathogenicity of O. tsutsugamushi.