Characterization of posttranslational modification of 19 kDa protein expressed by Mycobacterium avium subspecies paratuberculosis

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and has recently been linked to Crohn's disease in humans. To generate an effective vaccine against MAP, it is necessary to identify MAP antigens that trigger protective immunity. Unfortunately, not much is known about MAP proteins despite decades of research. We have previously shown that a 4.8 kb insert from MAP will produce a 16 kDa recombinant protein when expressed in Escherichia coli and 19 kDa recombinant protein when expressed in M smegmatis ( smeg 19K). The difference of 3 kDa in size of these expressed proteins may be related to posttranslational modificatjons that occur in Mycobacterium species. We hypothesized that smeg19K is a lipoglycoprotein since blast analysis revealed approximately 76 % amino acid identity between the MAP 19 kDa protein and a known lipoglycoprotein, the 19 kDa protein of M tuberculosis. This prediction was confirmed following positive staining of smeg19K with Sudan Black 4B, a postelectrophoresis dye used to stain for lipids. Smeg 19K has also stained positively for glycosylation with the lectin concavalin A, a highly specific stain for mannose residues. As expected, treatment with tunicamycin (an antibiotic known to inhibit N-glycosylation) and treatment with deglycosylation assay (non-specific for mannose ), showed no reduction in size of 19 kDa glycolipoproteins. Since covalent modification of proteins with acyl or glycosyl moieties alter immunogenicity and/or pathogenicity, the study here provides foundation for future experiments regarding the antigenicity of MAP 19 kDa lipoglycoprotein and its role in disease pathogenicity.

Notes

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Thesis Completion

2008

Semester

Spring

Advisor

Naser, Saleh A.

Degree

Bachelor of Science (B.S.)

College

Burnett College of Biomedical Sciences

Degree Program

Molecular Biology and Microbiology

Subjects

Arts and Sciences -- Dissertations, Academic;Dissertations, Academic -- Arts and Sciences

Format

Print

Identifier

DP0022312

Language

English

Access Status

Open Access

Length of Campus-only Access

None

Document Type

Honors in the Major Thesis

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