Recombination of Fragments of Human Growth Hormone: Altered Activity Profile of the Recombinant Molecule*

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Abstract
In previous work, we demonstrated that monomeric human GH (hGH) digested with thrombin can be resolved after reduction and S-carbamidomethylation into two peptides, one comprising residues 1–134 (here designated 1–134T) and the carboxyl-terminal peptide 135–191 (designated 135–191T). In the present study, the noncovalent recombination of these peptides is described. S-carbamidomethylated peptides 1–134 (1–134C) and 141–191 (141–191C) have also been prepared from a more acidic form of hGH (C-type) isolated during the preparation of hGH monomer by chromatography on diethylaminoethyl-cellulose. The recombination of these peptides has also been studied. Finally, peptide 42–134 has been produced by digestion of peptide 1–134T with human plasmin. Various attempts to recombine this peptide with peptides 135–191T or 141–191C were unsuccessful. Noncovalent recombination of the peptides was achieved by dissolving equimolar amounts of the materials in 0.5% ammonium bicarbonate solution containing 6 M guanidine-HCl or 6 M urea and dialyzing the mixture against the solvent. The recombinant was recovered by gel filtration of the peptide mixture. This procedure was used on a nanomolar scale by employing 125I-labeled amino-terminal peptide and the carboxyl-terminal peptide labeled with 14C by alkylating its reduced precursor with [l-14C]iodoacetamide. Recombinants of unlabeled peptides 1– 134T and 135–191T were also produced in micromolar quantities in about 70% yield. The recombinants of peptides 1–134T and 135–191T were about 35% as active as native hGH in the weight gain test in hypophysectomized rats. Their diabetogenic activity in the ob/ ob mouse was equivalent to that of native hGH, but their insulinlike activity (stimulating glucose oxidation in vitroin adipose tissue of hypophysectomized rats) was only 20% that of the native hormone. The recombinant preparations were not more than 20% as effective as native hGH in competing with [125I] iodo-ovine PRL for binding to liver cell membranes from female rats. They were, however, nearly equipotent with native hGH in the RIA for hGH. The profile of biological activity of the recombinant preparations is therefore quite different from that of native hGH but is reminiscent of that of the reduced and alkylated thrombin digests from which their constituent peptides were derived. The reason for the alteration in biological profile is the objective of further study.