An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described.
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.
Insulin‐like growth factor (IGF) binding to the type 1 IGF receptor (IGF1R) elicits mitogenic effects, promotion of differentiation and protection from apoptosis. This study has systematically measured IGF1R binding affinities of IGF‐I, IGF‐II and 14 IGF analogues to a recombinant high‐affinity form of the IGF1R using BIAcore technology. The analogues assessed could be divided into two groups: (a) those designed to investigate binding of IGF‐binding protein, which exhibited IGF1R‐binding affinities similar to those of IGF‐I or IGF‐II; (b) those generated to probe IGF1R interactions with greatly reduced IGF1R‐binding affinities. The relative binding affinities of IGF‐I analogues and IGF‐I for the IGF1R determined by BIAcore analysis agreed closely with existing data from receptor‐binding assays using cells or tissue membranes, demonstrating that BIAcore technology is a powerful tool for measuring affinities of IGFs for IGF1R. In parallel studies, IGF1R‐binding affinities were related to ability to protect against serum withdrawal‐induced apoptosis in three different assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromocytoma cell line, PC12. In this model system, IGF‐I and IGF‐II at low nanomolar concentrations are able to prevent apoptosis completely. We conclude that ability to protect against apoptosis is directly related to ability to bind the IGF1R.
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