Cloning, expression and purification of a partial outer membrane protein (Omp) from Candidatus Liberibacter asiaticus causing greening disease of citrus

H.Y. Lau, H. Habibuddin and C.S. Tan

Abstract

Citrus greening is one of the most serious diseases affecting citrus cultivation in many countries including Malaysia. The disease is caused by a non-cultured, phloem-restricted bacterium-like organism, Candidatus Liberibacter asiaticus. The low concentration of the organism in infected plants makes it difficult to obtain sufficient amounts of the pathogen to be used as antigen in the production of its antibodies. This study was carried out to produce recombinant protein from the partial outer membrane protein (Omp) that could be used as the antigen during polyclonal antibody production. The gene encoding the Omp of the organism was amplified using total DNA extracted from disease infected citrus midribs. The amplified PCR fragment of length 877 bp was purified, restriction enzyme digested and ligated into a bacterial expression plasmid vector, pRSET B. After the construct was verified by restriction enzyme and DNA sequencing, the bacterial expression was carried out using E. coli JM109 (DE3) and the expression of recombinant Omp was induced with isopropyl thiogalactoside (IPTG). The expected 30 kDa expressed protein was estimated by migration in 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant Omp was purified by nickel-chelating resin under denaturing conditions. This protein will be used in our subsequent studies to obtain antibodies against it.

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