Characterization and purification of proteins suitable for the production of antibodies against ‘Ca. Liberibacter asiaticus’

• Published in: Protein expression and purification 2017-11, Vol.139, p.36-42
• Main Authors: Liu, Huawei, Atta, Sagheer, Hartung, John S.
• Format: Article
• Language: English
• Subjects: Alphaproteobacteria - genetics; Antigens; Antigens, Bacterial - chemistry; Antigens, Bacterial - genetics; Antigens, Bacterial - immunology; Antigens, Bacterial - metabolism; Cloning, Molecular; Epitopes - chemistry; Epitopes - genetics; Epitopes - immunology; Epitopes - metabolism; Escherichia coli - genetics; HLB; Huanglongbing; Plant Diseases - microbiology; Protein purification; Protein structure; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2017.07.010

The citrus disease huanglongbing (HLB), which is caused by ‘Candidatus Liberibacter asiaticus’ (CaLas), is one of the most devastating pathogens of citrus, and with no effective method of control, poses a serious threat to citrus production throughout the world. In a previous study we described the production of single chain antibodies against several CaLas proteins that provide the basis for efficient and accurate detection of CaLas in citrus tissues. The isolation of a sufficient amount of purified antigen is a key step in the production of functional antibodies. The current report details purification procedures for six protein antigens used to select recombinant and produce polyclonal antibodies. These proteins include a flagellar biosynthesis protein (FlhA), a dinucleoside polyphosphate hydrolase (InvA), a portion of the major outer membrane protein (OmpA), a component of type IV pilus (CapB), the polysialic acid capsule expression protein (KpsA) and the outer membrane efflux protein (TolC). Results of purification under completely native or denatured conditions were not satisfactory. Therefore different hybrid purification conditions were optimized for each of the different proteins. The results of bioinformatic analysis also indicated that the six proteins contained a great diversity of potential antigenic epitopes, which varied in number, and that the antigenic clusters were not uniformly distributed throughout the proteins. The purified proteins are useful for the development of highly specific antibodies capable of differentiating specific strains of Liberibacter. •Proteins were identified from genome sequence data of ‘Ca. Liberibacter asiaticus’.•Bioinformatics was used to predict antigenic regions.•Antigenic regions were cloned in E. coli, expressed and purified.•Purification protocols were optimized for each antigenic protein fragment.•Antigenic domains were compared across the ‘Ca. Liberibacter sp.’.