Total chemical synthesis of ubiquitin using BOP reagent: biochemical and immunochemical properties of the purified synthetic product.
The complete ubiquitin molecule (76 residues) has been synthesized by the solid-phase method of Merrifield by using BOP as the coupling reagent. The crude product was purified by gel filtration, middle-pressure liquid chromatography and ion-exchange chromatography. Seven mg of a ca. 95% pure peptide was finally obtained; the overall yield of the synthesis was 1%. The final product was controlled by amino acid analysis and sequencing, HPLC and FAB-Mass spectrometry. Synthetic ubiquitin was found to be antigenically active in immunoblotting experiments and in an enzyme-linked immunosorbent assay with anti-ubiquitin antibodies as well as with anti-ubiquitin autoantibodies from autoimmune patients. Its activity was controlled in a conjugation enzymatic system and was found similar to that of commercial ubiquitin. The successful total synthesis of ubiquitin opens the way to the preparation of various stable analogs that should be useful for studying the intracellular metabolism of this molecular and its involvement in the protein degradation pathway.
Briand
J P
JP
Departement d'Immunochimie, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
Van Dorsselaer
A
A
Raboy
B
B
Muller
S
S
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014452:Ubiquitins / Q000032:analysis / Q000138:chemical synthesis* / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D007202:Indicators and Reagents; D009943:Organophosphorus Compounds; D014452:Ubiquitins; C031032:benzotriazol-1-yloxy-tris(dimethylamino)phosphonium; D001616:beta-Galactosidase