Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide.
The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni2+-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni2+-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.
Julia Scerbo
M
M
Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA Córdoba, Argentina.
Bibolini
Mario J
MJ
Barra
José L
JL
Roth
German A
GA
Monferran
Clara G
CG
D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000276:immunology* / Q000627:therapeutic use; D004768:Enterotoxins / Q000096:biosynthesis* / Q000276:immunology* / Q000627:therapeutic use; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000276:immunology* / Q000627:therapeutic use; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006968:Hypersensitivity, Delayed / Q000188:drug therapy / Q000276:immunology; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008297:Male; D010455:Peptides / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use; D017510:Protein Folding; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology* / Q000627:therapeutic use; D016704:Synapsins / Q000096:biosynthesis* / Q000276:immunology* / Q000627:therapeutic use
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D016704:Synapsins; C027538:heat-labile enterotoxin, E coli