ras p21 deletion mutants and monoclonal antibodies as tools for localization of regions relevant to p21 function

Abstract

Deletion mutants of the viral Harvey ras oncogene were generated by removing different lengths of the gene from either the amino or the carboxyl terminus. The deletion mutants, ras p21 expressed in Escherichia coli, yielded proteins of approximately 8, 10, 12, 14, 17, 18, 19, and 20 kDa. These proteins were utilized to identify epitopes recognized by a series of recently generated monoclonal antibodies as well as some previously reported monoclonal antibodies. Monoclonal antibodies that inhibited GTP binding, a major biochemical activity of the p21 protein, recognized two major regions of the protein. These regions were localized from amino acids 5 to 69 and 107 to 164, respectively, and were separated by another stretch from residues 70 to 106, whose antigenic determinants were not directly involved in GTP binding. Thus, the mapping of epitopes within the p21 molecule recognized by monoclonal antibodies has made it possible to localize important functional regions within the ras p21 molecule.