Gene cloning and expression of Xanthomonas citri subsp. citri pilin

Citrus bacterial canker is a disease caused by Xanthomonas citri subsp. citri (Xcc). The bacterial type III secretion system (TTSS) consist of an extracellular filamentous structure mediating transfer of bacterial proteins into the plant cytosols. The main part of the pilus is pilin protein (HrpE) that is encoded by the HrpE gene. Production of specific antibodies against the pilin protein can be implemented for detection of infected plants as well as to develop a source of genetic resistance against disease. Inhibition of HrpE protein through antibody therapy leads to suppression of disease in the plant. The objective of the present study was to isolate, clone and express the HrpE gene. To do this, the HrpE gene was amplified by PCR using gene-specific primers and cloned to the pTZ57R/T vector. Then, it was cloned to the pET28a() bacterial expression vector and expressed in the E. coli strain Rosetta as host. The protein production procedure was optimized for incubation time and temperature as well as the concentration of inducer. The greatest amount of the recombinant protein was yielded at 1 mM IPTG at 30° C for 16 h. Purification of HrpE recombinant protein was done via metal affinity chromatography. The results of both SDS-PAGE and Western blotting assays confirmed the expression accuracy and purity of recombinant pilin protein. The recombinant protein can be used as an antigen to develop monoclonal and polyclonal antibodies.