Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu 3, following their expression in Escherichia coli as fusion proteins

Abstract

The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.

Interaction of insulin-like growth factor (IGF)-I and -II with IGF binding protein-2: mapping the binding surfaces by nuclear magnetic resonance

“…Recombinant human IGF was expressed and purified from Escherichia coli JM101 cells [F' traD36 proAB laclqZ M15]) as described previously (King et al 1992, Francis et al 1993. Briefly, E. coli containing the IGF expression vectors Figure 1 Amino acid sequences of human IGF-I and -II.…”

“…pGH(1-11)-VN-IGF-I (Francis et al 1993), pGH(1-11)-VNPAPH-IGF-II (Lien et al 2001) or pGH(1-11)-VNFAHY-IGF-II (Francis et al 1993) were maintained in minimal medium with 60 mM ( 15 NH 4 ) 2 SO 4 as the sole source of nitrogen in 2 l bioreactors (Applikon Bioreactor, Schiedam, Holland) as described by King et al (1992) 4 . Protein was recovered from inclusion bodies by resuspending in 8 M guanidine HCl followed by desalting on the fast protein liquid chromatograph using a 50 200 mm column packed with Sephadex G-25 M (Pharmacia).…”

Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency

“…Recently, we described an efficient expression system in Escherichia coli for IGF-I analogues as fusion peptides (King, Wells, Krieg et al 1992). …”

Purification and molecular characterization of recombinant rat betacellulin

“…The mature form of rat BTC (Asp 32 -Tyr 111 ) was expressed as a recombinant fusion protein containing the first 46 amino-terminal amino acids of porcine growth hormone (pGH) (King et al 1992). A proteolytic cleavage site (Ile-Glu-Gly-Arg) was engineered downstream of the pGH fusion partner to allow for 'release' of rat BTC after digestion with Factor Xa (Fig.…”

“…The subsequent cDNA was used as a template for PCR with oligonucleotide primers, 5 ATC TAG GTT AAC ATC GAA GGT CGT GAT GGG AAC ACG ACC AGA ACC 3 (single underline, HpaI restriction site; double underline, nucleotide sequence encoding the Ile-Glu-Gly-Arg Factor Xa recognition site) and 5 CTA GAT AAG CTT TCA TCA GTA AAA CAG GTC CAC CTG 3 (single underline, HindIII restriction site, double underline stop codons). The resultant 282 bp PCR product was purified, digested with HpaI/HindIII and cloned into HpaI/HindIIIdigested 46-amino acid porcine growth hormone (pGH(1-46)) expression vector (King et al 1992) to generate pGH(1-46)-Ile-Glu-Gly-Arg-BTC (Fig. 1).…”