Recombinant hormones from fragments of human growth hormone and human placental lactogen.
Peptide fragments generated by limited plasmin digestion and reduction of human growth hormone (hGH) and human placental lactogen (hPL) were recombined to produce biologically active molecules consisting of the NH2-terminal two-thirds of one hormone molecule linked to the COOH-terminal one-third of the other. Plasmin-cleaved hPL and hGH each consist of an NH2-terminal fragment (1-134) connected to a COOH-terminal fragment (141-191) through a Cys53-Cys165 disulfide bond. The plasmin-cleaved hormones were reduced, the fragments isolated, and then recombined in equimolar quantities to prepared recombinant hormones. On sodium dodecyl sulfate disc gel electrophoresis the recombinants migrated as a single band in the position of native hormone when they were not reduced and as two bands in the positions of the fragments when they were reduced. This indicated that the recombinants had re-formed a disulfide bond. The derivatives were tested for immunologic activity in hPL and hGH radioimmunoassays, for lactogenic and growth-promoting activity by the radioreceptor assays using membranes from mammary gland and liver of lactating rabbits, and for secondary and tertiary structure by circular dichroism measurements in the near and far UV regions. The recombinant with NH2-terminal hGH had hGH immunologic activity, hGH structure and lactogenic and growth-promoting activity, while the hybrid with NH2-terminal hPL had hPL immunologic activity, hPL structure, and lactogenic activity alone. Therefore the immunologic activity, biologic activity, and structure of each of the covalently linked derivatives was characteristic of the hormone's NH2-terminal 1-134 fragment. The COOH-terminal 141-191 sequence maintains overall conformation but appears to have little, if any, role in determining biologic specificity.
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D004220:Disulfides; D005341:Fibrinolysin; D013006:Growth Hormone; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D010928:Placental Lactogen; D011487:Protein Conformation; D011863:Radioimmunoassay
D000596:Amino Acids; D004220:Disulfides; D010446:Peptide Fragments; D013006:Growth Hormone; D010928:Placental Lactogen; D005341:Fibrinolysin