title: Expression and purification of a mutant human growth hormone that is resistant to proteolytic cleavage by thrombin, plasmin and human plasma in vitro.
abstract: 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 Arg134 and Thr135 of hGH with Asp134 and Pro135 yielded a thrombin-resistant hGH mutant, and substitution of Arg134, Thr135 and Lys140 with Asp134, Pro135 and Ala140 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.
authors.affiliation: Department of Biochemistry, Mie University Faculty of Medicine, Japan.
authors.forename: K S
authors.initials: KS
authors.lastname: Alam
authors.forename: M
authors.initials: M
authors.lastname: Morimoto
authors.forename: H
authors.initials: H
authors.lastname: Yoshizato
authors.forename: T
authors.initials: T
authors.lastname: Fujikawa
authors.forename: K
authors.initials: K
authors.lastname: Furukawa
authors.forename: M
authors.initials: M
authors.lastname: Tanaka
authors.forename: K
authors.initials: K
authors.lastname: Nakashima
mesh_terms: D000818:Animals; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005341:Fibrinolysin / Q000378:metabolism*; D005822:Genetic Vectors; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D051381:Rats; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D018076:DNA, Complementary; D019382:Human Growth Hormone; D013917:Thrombin; D005341:Fibrinolysin