Insertion of myoglobin T-cell epitopes into the Escherichia coli alkaline phosphatase.
We are interested in antigen processing mechanisms of antigen-presenting cells, and to what extent the susceptibility of protein antigens to degradation contributes to immunogenicity. Understanding the biochemistry of antigen processing may be essential for reliable prediction of T-cell epitopes and for the design of vaccines that are optimized for T-cell priming. To examine possible effects of protein structural context on antigen presentation, we used genetic engineering techniques to insert helper T-cell epitopes derived from sperm whale myoglobin into surface loops of the highly stable Escherichia coli alkaline phosphatase, with the expectation that presentation of the myoglobin guest epitopes might vary with their position in the carrier protein. Levels of recombinant protein expression in E. coli cells and residual enzyme activity depended on the location of the guest peptides in the alkaline phosphatase carrier. Mutants with insertions between residues 189-190 of the carrier were recovered with yields and activities similar to the wild type protein; however, insertion of the same peptides at a second site, between residues 165-166, led to low recoveries and diminished phosphatase activities. Subcutaneous injection of mice with one of the purified recombinant proteins in complete Freund's adjuvant induced T cells that responded to in vitro challenge with myoglobin. The potential use of this system to dissect processing mechanisms is discussed.
Freimuth
P
P
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
Steinman
R M
RM
D000469:Alkaline Phosphatase / Q000235:genetics / Q000276:immunology; D000818:Animals; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D051379:Mice; D009211:Myoglobin / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics / Q000302:isolation & purification
D000939:Epitopes; D009211:Myoglobin; D014614:Vaccines, Synthetic; D000469:Alkaline Phosphatase