title: Synthesis and purification of a deleted human growth hormone, hGH delta 135-146: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities.
abstract: Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity. In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system. The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro. The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay. In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH. In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations. By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH. These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.
authors.affiliation: Department of Biochemistry, Mie University Faculty of Medicine, Japan.
authors.forename: K S
authors.initials: KS
authors.lastname: Alam
authors.forename: T
authors.initials: T
authors.lastname: Fujikawa
authors.forename: H
authors.initials: H
authors.lastname: Yoshizato
authors.forename: M
authors.initials: M
authors.lastname: Tanaka
authors.forename: K
authors.initials: K
authors.lastname: Nakashima
mesh_terms: D000818:Animals; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D005341:Fibrinolysin / Q000378:metabolism* / Q000494:pharmacology; D017353:Gene Deletion; D015870:Gene Expression; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007016:Hypophysectomy; D066298:In Vitro Techniques; D007334:Insulin-Like Growth Factor I / Q000235:genetics; D008223:Lymphoma; D008297:Male; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured; D015430:Weight Gain / Q000187:drug effects
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D007334:Insulin-Like Growth Factor I; D005341:Fibrinolysin