An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described.
1. Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) together with a truncated form of IGF-1 were purified to homogeneity from bovine colostrum. 2. Two forms of IGF-1 were totally resolved from IGF-2 in the purification by h.p.l.c. involving cation-exchange and reverse-phase columns. 3. The complete amino acid sequences for all three forms of IGF were determined. The sequence of bovine IGF-1 was found to be identical with that of human IGF-1, and that of the variant lacked the N-terminal tripeptide Gly-Pro-Glu (-3N:IGF-1). Bovine IGF-2 was found to differ in three residues of the C-domain compared with human IGF-2, with serine, isoleucine and asparagine substituted for alanine, valine and serine respectively at positions 32, 35 and 36. 4. Protein synthesis in L6 rat myoblasts was stimulated and protein degradation inhibited in a co-ordinate response with all three IGFs. The relative potency in both processes was -3N:IGF-1 greater than IGF-1 greater than IGF-2. A similar order of potency was obtained for the stimulation of DNA synthesis by -3N:IGF-1 and IGF-1. The approximately 10-fold effect on biological activity of removing the N-terminal tripeptide is unexpected in view of current information on IGF-1 structure and function.
We have investigated which region(s) of bovine insulin-like growth factor binding protein-2 (bIGFBP-2) interact with insulin-like growth factors (IGFs) using Cterminally truncated forms of bIGFBP-2. Initially to aid in mutant design, we defined the disulfide bonding pattern of bIGFBP-2 C-terminal region using enzymatic di-
Recombinant insulin-like growth factor-II (IGF-II) and two structural analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were produced to investigate the role of N-terminal residues in binding to IGF-binding proteins (IGFBPs) and hence the biological properties of the modified peptides. The growth factors were modelled on two previously characterized variants of IGF-I, des(1-3)IGF-I and [Arg3]-IGF-I, which both show substantially decreased binding to IGFBPs and were expressed as fusion proteins in Escherichia coli. The biological activities of the corresponding analogues of IGF-I and IGF-II were compared in rat L6 myoblasts and H35B hepatoma cells. In the L6-myoblast protein-synthesis assay, the IGF-II analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were slightly more potent than IGF-II but about 10-fold less potent than IGF-I and 100-fold less potent than the respective IGF-I analogues, des(1-3)IGF-I and [Arg3]IGF-I. In H35 hepatoma cells the anabolic response measured was the inhibition of protein breakdown, and the potency order was insulin >>> [Arg3]-IGF-I > des(1-3)IGF-I > [Arg6]-IGF-II > des(1-6)IGF-II > IGF-I > IGF-II. Binding of the IGFs and their analogues to the type 1 IGF receptor in L6 myoblasts and to the insulin receptor in H35 hepatoma cells did not fully explain the observed anabolic potency differences. Moreover, binding of all four analogues to the IGFBPs secreted by L6 myoblasts and H35B hepatoma cells was greatly decreased compared with the parent IGF. We conclude that the observed anabolic response to each IGF was determined by their relative binding to the competing cell receptor and IGFBP binding sites present.
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