title: Recombinant DNA approach for defining the primary structure of monoclonal antibody epitopes. The analysis of a conformation-specific antibody to myosin light chain 2.
abstract: A monoclonal antibody (MF5), capable of recognizing a divalent cation-induced conformational change in myosin light chain 2 (LC2f), has been used to screen a cDNA library constructed in the expression vector lambda gt11. A clone has been isolated that contains the whole coding sequence of this myosin subunit. The light chain was synthesized as a fusion peptide linked to beta-galactosidase by ten amino acids encoded in the 5' untranslated region of its mRNA. Seven imperfect repeats were identified in the 3' untranslated region of the mRNA. The amino acids conferring specificity on the MF 5 epitope were established by first determining the nucleotide sequence of shorter subclones that expressed the epitope and then eliminating those amino acid residues shared by cardiac myosin LC2, which was unreactive with this antibody. The epitope, which becomes accessible to MF 5 upon removal of bound divalent cations, resides at the junction between the first alpha-helical domain and the metal binding site. Theoretically, this approach can be used to define the primary structure of most protein epitopes.
authors.forename: F C
authors.initials: FC
authors.lastname: Reinach
authors.forename: D A
authors.initials: DA
authors.lastname: Fischman
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001345:Autoradiography; D010582:Bacteriophage lambda; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005822:Genetic Vectors; D009218:Myosins; D012333:RNA, Messenger
chemical_list: D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D000939:Epitopes; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes; D009218:Myosins