EXPRESSION AND PURIFICATION OF A MUTANT HUMAN GROWTH-HORMONE THAT IS RESISTANT TO PROTEOLYTIC CLEAVAGE BY THROMBIN, PLASMIN AND HUMAN PLASMA IN-VITRO

The region having a sequence from amino acid 134 to 150 in human growt h hormone (hGH) is known to be cleaved by proteases in human plasma, p lasmin and thrombin. In this study, oligonucleotide primer-directed mu tagenesis was used to produce recombinant mutant hGHs resistant to lim ited proteolysis by these proteases. Substitution of Arg(134) and Thr( 135) of hGH with Asp(134) and Pro(135) yielded a thrombin-resistant hG H 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. T he latter mutant hGH was also insensitive to in vitro proteolysis by h uman plasma incubated for 7 days. These alterations in amino acid resi dues of hGH did not disrupt its biological conformation and retained f ull growth promoting activities on rat Nb2 cells and human T-47D breas t cancer cells. (C) 1998 Elsevier Science B.V. All rights reserved.