RESUMO
A fusion protein (FP) comprised of the RNase A S-peptide and human epidermal growth factor was shown to form a stable noncovalent catalytically active complex with the RNase A S-protein at a stoichiometric ratio 1:1 with Kdiss = 5.0 x 10(-7) M. The S-protein complex with FP exhibits the pyrimidine specificity toward substrates in both reactions catalyzed by RNase S, transesterification and hydrolysis. The fusion protein can be determined specifically and quantitatively in the presence of S-protein by RNase activity assays. The possibility of effective purification of S-peptide-containing proteins by affinity chromatography on an S-protein-Sepharose column has been demonstrated.
Assuntos
Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Ribonuclease Pancreático/metabolismo , Ribonucleases/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptídeos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease Pancreático/química , Ribonuclease Pancreático/genética , Ribonucleases/química , TermodinâmicaRESUMO
The fused polypeptides of human epidermal growth factor and one or two S-peptide of RNase A was shown to form stoichiometric (1:1) strong noncovalent and enzymatically active complexes with S-protein of RNase A. The dissociation constants for these complexes were found to be 5.0 x 10(-7) M and 1.1 x 10(-7) M. The complexes of polypeptides with S-protein were capable to hydrolyze ribopolynucleotides, and pyrimidine-2',3'-cyclophosphates specifically, like RNase S'. A possibility was shown of effective purification of the S-peptide-containing polypeptides by affinity chromatography in which S-protein is immobilized on solid supports.