Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli

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The development of a homologous RIA for chicken insulin-like growth factor-II (cIGF-II) and its application to investigate the developmental changes in IGF-II in the chicken and turkey are described. A double-antibody RIA has been developed using recombinantly derived cIGF-II as antigen, radiolabelled tracer and standard. Serial dilutions of chicken and turkey plasma were parallel to serial dilutions of cIGF-II standard. We have also established that acid/ethanol extraction of chicken and turkey plasma reduced possible interference of insulin-like growth factor-binding proteins in the RIA. Consumption of a low-protein diet by male chickens lowered plasma IGF-I twofold, whereas IGF-II levels were unchanged. Food withdrawal evoked an increase in circulating IGF-II, while IGF-I levels were reduced. Refeeding returned both growth factors to normal circulating concentrations. During chick embryo incubation, plasma IGF-II levels were tenfold higher than those of IGF-I. In the turkey embryo, plasma IGF-II concentrations were higher than those of IGF-I. During the post-hatch period, IGF-II levels declined with age in chickens. In the growing turkey, IGF-II levels were consistently higher than IGF-I levels. The application of the homologous RIA to monitor plasma levels during embryonic development and posthatch growth in avian species will provide more accurate comparisons of results from studies on the role of IGF-II in growth and metabolism of domestic birds.

Section: Discussionmentioning

confidence: 98%

“…More is known about the function of these multifunctional proteins in mammals than in avian species. Chicken IGF-I (cIGF-I) is more closely related to its mammalian counterpart in terms of amino acid sequence ) than is chicken IGF-II (cIGF-II) (Upton et al 1995). Moreover, cIGF-II differs in 12 amino acids from human IGF-II.…”

Section: Introductionmentioning

confidence: 99%

Assessment of developmental changes in chicken and turkey insulin-like growth factor-II by homologous radioimmunoassay

McMurtry¹,

Rosebrough²,

Brocht³

et al. 1998

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“…Quantification of kIGF-I and kIGF-II was performed as described by Upton et al (1995) using the calculated absorption coefficient 33·38 mol 1 cm 1 for kIGF-I. As the complete sequence of kIGF-II was not determined the absorption coefficient calculated for hIGF-II (31·701 mol 1 cm 1 ) was used.…”

Section: Quantification and Sequencingmentioning

confidence: 99%

Purification, amino acid sequence and characterisation of kangaroo IGF-I

Yandell

Francis²,

Wheldrake³

et al. 1998

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Insulin-like growth factor-I (IGF-I) and IGF-II have been purified to homogeneity from kangaroo (Macropus fuliginosus) serum, thus this represents the first report of the purification, sequencing and characterisation of marsupial IGFs. N-Terminal protein sequencing reveals that there are six amino acid differences between kangaroo and human IGF-I. Kangaroo IGF-II has been partially sequenced and no differences were found between human and kangaroo IGF-II in the 53 residues identified. Thus the IGFs appear to be remarkably structurally conserved during mammalian radiation. In addition, in vitro characterisation of kangaroo IGF-I demonstrated that the functional properties of human, kangaroo and chicken IGF-I are very similar. In an assay measuring the ability of the proteins to stimulate protein synthesis in rat L6 myoblasts, all IGF-I proteins were found to be equally potent. The ability of all three proteins to compete for binding with radiolabelled human IGF-I to type-1 IGF receptors in L6 myoblasts and in Sminthopsis crassicaudata transformed lung fibroblasts, a marsupial cell line, was comparable. Furthermore, kangaroo and human IGF-I react equally in a human IGF-I RIA using a human reference standard, radiolabelled human IGF-I and a polyclonal antibody raised against recombinant human IGF-I. This study indicates that not only is the primary structure of eutherian and metatherian IGF-I conserved, but also the proteins appear to be functionally similar.

“…Purity was assessed by N-terminal protein sequencing and quantitation was performed as described by Upton et al (1995) using the calculated absorption coefficient of 31·69 g/l per cm at 214 nm.…”

Section: Methodsmentioning

confidence: 99%

Kangaroo IGF-II is structurally and functionally similar to the human [Ser29]-IGF-II variant

Yandell¹,

Francis²,

Wheldrake³

et al. 1999

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Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser 29 ]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein.The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser 29 ]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cationindependent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser 29 ]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.