Synthesis and purification of a deleted human growth hormone, hGHΔ135-146:: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities

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. (C) 2000 Elsevier Science B.V. All rights reserved.