Expression and purification of a mutant human growth hormone that isresistant to proteolytic cleavage by thrombin, plasmin and human plasma invitro

摘要

The region having a sequence from amino acid 134 to 150 in human growth hormone (hGH) is known to be cleaved by proteases in human plasma, plasmin and thrombin. In this study, oligonucleotide primer-directed mutagenesis was used to produce recombinant mutant hGHs resistant to limited proteolysis by these proteases. Substitution of Arg(134) and Thr(135) of hGH with Asp(134) and Pro(135) yielded a thrombin-resistant hGH mutant: and substitution of Arg(134), Thr(135) and Lys(140) With Asp(134), Pro(135) and Ala(140) yielded a plasmin-resistant hGH mutant. The latter mutant hGH was also insensitive to in vitro proteolysis by human plasma incubated for 7 days. These alterations in amino acid residues of hGH did not disrupt its biological conformation and retained full growth promoting activities on rat Nb2 cells and human T-47D breast cancer cells.