Binding protein, radioreceptor and biological activities of recombinant methionyl insulin-like growth factor-I variants

Abstract

Reversed-phase chromatography (RFC) was used to resolve two variants of recombinant amino terminal methionyl residue (TV-Met) insulin-like growth factor-I (IGF-I) with the same amino acid constitution but different disulphide linkages. Following radioiodination, equilibration with plasma and size exclusion chromatography at neutral pH the major form on RPC (approximate abundance 60%) demonstrated > 80% binding to 150 kDa and 40–50 kDa IGF binding proteins. This peptide has the RPC elution characteristics and disulphide assignment (Cys 6-Cys 48, Cys 18-Cys 61, Cys 47-Cys 52) of authentic IGF-I (termed TV-Met IGF-I peak 2 peptide). By contrast, a second peptide (approximate abundance 25%) with mismatched disulphides (Cys 6-Cys 47, Cys 18-Cys 61, Cys 48-Cys 52; N-Met IGF-I peak 1 peptide) demonstrated < 15% binding under similar conditions. Potency of the peptides was investigated in competitive IGF-I plasma binding protein and L6 myoblast radioreceptor assays. The peak 2 peptide proved equipotent to purified ovine plasma IGF-I in each system but by contrast the peak 1 peptide was 40-fold and 200-fold less potent in the binding protein and radioreceptor assays respectively. Biological potency was examined in a non-competitive assay based on incorporation of [ 3H]leucine into confluent cultures of L6 myoblasts. In this system the N-Met IGF-I peak 1 peptide proved 15-fold less potent than the peak 2 peptide with correct disulphide linkages. Refolding variants may prove useful in establishing structure/function relationships for IGF-I.