AMINO ACID AT THE SUPPRESSION SITE IN RABBIT BETA-GLOBIN READTHROUGH PROTEIN

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Rabbit beta-globin readthrough protein is the only known naturally occurring readthrough protein in higher eukaryotes which does not involve a viral system. Since suppressor tRNAs have been used in gene therapy experiments and have been implicated in inhibiting viral expression, it is important to characterize the nonsense suppressor tRNA involved in the expression of this unique protein. Experiments were devised, therefore, to isolate this protein from rabbit reticulocytes and to identify the amino acid at the readthrough site. Specific antibodies against this protein were prepared by synthesizing a 22 amino acid peptide which corresponds to the readthrough portion of the beta-globin readthrough protein, coupling the peptide to the KLH protein and injecting the conjugated protein into a sheep. Specific antibodies were produced which were purified and used to isolate the readthrough protein. The protein was purified to homogeneity by electrophoresis on a polyacrylamide gel and the purified product is presently being sequenced through the readthrough site.