- Email: [email protected]
In vitro characterization and in vivo clearance of recombinant barramundi (Lates calcarifer) IGF-I
Aquaculture 177 Ž1999. 153–160
In vitro characterization and in vivo clearance of recombinant barramundi žLates calcarifer / IGF-I Brian G. Degger a,b, Neil Richardson b, Chris Collet b, F. John Ballard a , Zee Upton a,) a
CooperatiÕe Research Centre for Tissue Growth and Repair, School of Biological Sciences, GPO Box 2100, Adelaide, SA 5001, Australia b School of Life Sciences, Queensland UniÕersity of Technology, Brisbane, QLD, Australia Accepted 1 October 1998
Abstract Little is known about fish insulin-like growth factors ŽIGFs. as only small amounts have been isolated from native sources or indeed produced recombinantly. This report describes the production of milligram quantities of recombinant barramundi IGF-I ŽbIGF-I. and its subsequent characterization. Recombinant bIGF-I was produced in Escherichia coli using a gene fusion system similar to that previously described for the production of other non-mammalian IGFs. Recombinant bIGF-I was similar to human IGF-I ŽhIGF-I. in stimulating protein synthesis and in competing for binding of labelled hIGF-I to IGF receptors whether tested in rat myoblasts or in salmon embryo fibroblasts. However, recombinant bIGF-I differed from its human counterpart in its affinity for a polyclonal antibody raised against hIGF-I, with at least 200-fold more bIGF-I required to obtain 50% displacement of labelled hIGF-I from the antibody. Hence, the recombinant protein will be essential for developing a specific homologous immunoassay for measuring IGF-I concentrations in barramundi during growth and development. In addition, studies investigating the clearance of labelled bIGF-I and hIGF-I in vivo reveal that the human protein is cleared from the circulation of juvenile barramundi almost twice as fast as the barramundi protein, thus providing the first in vivo evidence that there are functional differences between fish and human IGF-Is. Neutral gel chromatography of serum from the clearance study suggest that this is due to differences in the affinities of the labelled human and fish IGF-I for the IGFBPs present in barramundi. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Barramundi; Lates calcarifer; IGF-I
)
Corresponding author. Tel.: q61-8-8201-5248; Fax: q61-8-8201-3015; E-mail: [email protected] 0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 0 7 5 - 7
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1. Introduction Compared to the situation in mammals, little is known about fish insulin-like growth factors ŽIGFs. and their role in growth and development as only small amounts have been available for such experiments ŽMoriyama et al., 1993, 1995.. However, there is growing evidence from heterologous studies to suggest that IGF-I is also important in fish. Indeed, a number of in vivo studies have shown that elements of the growth hormone–IGF axis are found in fish and that the growth-promoting actions of IGF-I observed in mammals are conserved in fish ŽMcCormick et al., 1991; Moriyama, 1995.. In an attempt to determine whether similar results would be obtained if these studies were undertaken using homologous reagents, recombinant barramundi Ž Lates calcarifer . IGF-I ŽbIGF-I. has been produced using procedures similar to those previously described for the production of milligram quantities of other IGFs ŽUpton et al., 1992, 1996.. While DNA sequences have been reported for a number of fish IGF-Is, production of barramundi IGF-I was pursued due to the importance of this species in aquaculture in the tropical and sub-tropical regions of the Indo-Pacific and Australia. Thus, the aim of the present study was to produce recombinant bIGF-I in quantities to facilitate not only homologous in vitro, but also in vivo investigations.
2. Materials and methods 2.1. Generation of expression construct and fermentation Using site-directed mutagenesis ŽMutagene Kit, BioRad, South Richmond, CA, USA. and a synthetic gene for chicken IGF-I as the starting template, a construct for expression of recombinant barramundi IGF-I ŽbIGF-I. was engineered. The DNA sequence information for the protein was generously provided by Dr. A. Kinhult ŽSchool of Life Sciences, Queensland University of Technology, Brisbane, QLD, Australia.. The engineered construct features codons selected for maximal expression in Escherichia coli. In addition, the expression vector was designed such that when E. coli JM101 Ž lacI q . cells transformed with the construct are fermented, the IGF is expressed as a fusion protein in inclusion bodies ŽUpton et al., 1992.. Fermentation and the subsequent homogenization and centrifugation of the broth to collect the inclusion bodies were performed as described in Upton et al. Ž1992, 1996.. 2.2. Purification of barramundi IGF-I from inclusion bodies Inclusion bodies containing the bIGF-I fusion protein were processed as described by Upton et al. Ž1996. with the exceptions that the fusion protein was refolded in 4 M urea, while the release of the bIGF-I from the fusion partner with hydroxylamine was performed in the presence of 2.5 M urea. Methods for quantitation and chemical analysis of the recombinant protein are also reported in Upton et al. Ž1996..
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2.3. In Õitro characterization The functional properties of recombinant barramundi IGF-I were compared to human IGF-I ŽhIGF-I; GroPep, Adelaide, SA, Australia. in a number of in vitro characterization systems. Stimulation of protein synthesis in L6 rat myoblasts was performed according to Ballard et al. Ž1986.. Assessment of binding to type-1 IGF receptors in cultured cells was performed according to Ross et al. Ž1989., while binding to the type-2 IGF receptors in ovine placental membranes was measured as described by Read et al. Ž1986.. This same publication describes the method used for comparing the cross-reactivities of barramundi and human IGF-I in a radioimmunoassay using a polyclonal antibody prepared against hIGF-I ŽGroPep, Adelaide, SA, Australia.. Radiolabels used in the in vitro characterization assays and in the in vivo clearance study described below were prepared using the chloramine-T procedure ŽFrancis et al., 1988. and were used as indicated in the figures. 2.4. In ÕiÕo clearance of labelled IGF-I in fish Clearance of labelled human and barramundi IGF-I from the circulation of juvenile barramundi Ž90–150 g. was evaluated by administering a bolus injection of the radiolabel intraperitoneally at t s 0 Ž1500 cpmrg body weight. and then sacrificing five fish at each time point Ž1, 2, 4, 6, 10 and 24 h., with the collection of individual serum samples. Determination of TCA-precipitable radioactivity and neutral gel exclusion chromatography of serum samples were performed as described in McMurtry et al. Ž1996..
3. Results 3.1. Fermentation and downstream processing A 10-l fermentation of E. coli transformed with the construct for expression of the bIGF-I fusion protein yielded 55 g of inclusion bodies. Twenty-five grams of the inclusion bodies were processed, resulting in a yield of 20 mg of recombinant bIGF-I. The growth factor recovered from the final step of the downstream processing eluted as a single peak on an analytical high-performance liquid chromatography ŽHPLC. and migrated as a single band with the expected size of approximately 7.3 kDa on a high density sodium dodecyl sulfate ŽSDS.-polyacrylamide gel run under reducing conditions ŽFig. 1.. N-terminal peptide sequencing indicated that the protein had the correct N-terminal sequence and was at least 94% pure based on the average recovery of the major PTH-amino acid from the first five cycles of the Edman degradation. Mass spectrometry indicated that the majority of the protein had a mass 15 Da higher than expected, which, we have now established, is due to the conservative substitution of Ala for Gly 19 . This substitution is the result of a single nucleotide change in the transformed E. coli clone that was fermented.
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Fig. 1. Ža. Analysis of pure recombinant bIGF-I Žlane 2. electrophoresed on a high-density SDS-polyacrylamide gel under reducing conditions. Arrows indicate the size of the molecular mass markers Žlane 1.. Žb. Analysis of pure recombinant bIGF-I on a microbore C4 reverse-phase HPLC column. Elution of protein, monitored as absorbance at 215 nm, was achieved by a gradient of acetonitrile Ždotted line. in 0.1% Žvrv. trifluoroacetic acid.
3.2. In Õitro characterization of bIGF-I The functional properties of recombinant bIGF-I were compared with hIGF-I in a number of in vitro characterization systems. Thus, the abilities of the two peptides to compete for binding of radiolabelled IGF-I to type-1 IGF receptors in CHSE-214 salmon embryo fibroblasts were similar, with half-maximal effects observed at 14.2 " 3.5 ngr0.5 ml and 15.0 " 2.3 ngr0.5 ml for bIGF-I and hIGF-I, respectively. Likewise, the two proteins competed for binding to the type-1 receptors on L6 rat myoblasts to a
Fig. 2. Effects of IGF-Is on binding of labelled hIGF-I to Ža. L6 rat myoblasts and Žb. CHSE-214 salmon embryo fibroblasts. The proteins tested were hIGF-I Ž'. and bIGF-I ŽI.. Values are the means of triplicate determinations at each peptide concentration. ŽSEM values are indicated by vertical bars where they are larger than the symbols..
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Fig. 3. Effects of IGFs on binding of labelled hIGF-II to bovine placental membranes. The proteins tested were hIGF-I ŽB., bIGF-I ŽI. and hIGF-II Žv .. Values are the means of triplicate determinations at each peptide concentration. ŽSEM values are indicated by vertical bars where they are larger than the symbols..
similar extent with half-maximal competition at 2.3 " 0.3 ngr0.5 ml for bIGF-I and 2.0 " 0.2 ngr0.5 ml for hIGF-I ŽFig. 2.. The proteins were also essentially equipotent in their abilities to stimulate protein synthesis in rat myoblasts Žresults not shown.. The human and barramundi proteins differed, however, in their abilities to compete for binding of labelled hIGF-II to ovine placental membranes. While half-maximal effects were not determined for this assay, bIGF-I clearly showed greater ability to compete for binding of the radiolabel than did hIGF-I ŽFig. 3..
Fig. 4. Radioimmunoassay of IGFs using a polyclonal antiserum raised against hIGF-I and hIGF-I as radiolabel. The proteins tested were hIGF-I ŽB. and bIGF-I ŽI.. Values are the means of triplicate determinations at each peptide concentration. ŽSEM values are indicated by vertical bars where they are larger than the symbols..
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Fig. 5. Clearance of labelled IGFs in vivo: percent of intact labelled protein remaining in the circulation over time. The proteins examined were labelled hIGF-I ŽB. and labelled bIGF-I ŽI.. Each point represents the mean value from TCA precipitations on sera from five individual fish at each time point. ŽSEM values are indicated by vertical bars where they are larger than the symbols..
The cross-reactivities of bIGF-I and hIGF-I were compared in an immunoassay using a polyclonal antibody raised against hIGF-I and hIGF-I as the radiolabel. Barramundi IGF-I was 200-fold less effective than human IGF-I at competing for binding to this antibody ŽFig. 4.. 3.3. In ÕiÕo clearance of labelled IGF-I in barramundi Examination of the clearance of radiolabelled hIGF-I and bIGF-I from the circulation of juvenile barramundi revealed interesting differences between the two proteins. Labelled hIGF-I is cleared from the circulation of juvenile barramundi more rapidly than labelled bIGF-I. Indeed, the human protein appears to be removed from the circulation almost twice as fast as bIGF-I ŽFig. 5.. Neutral gel chromatography of serum from the fish in this study suggests that the differences in the clearance of hIGF-I and bIGF-I are due to differences in the affinities for the IGF-binding proteins present in barramundi serum Žresults not shown..
4. Discussion In this report, the production of milligram quantities of recombinant bIGF-I from E. coli and the subsequent characterization of this protein compared with its human counterpart are described. In vitro characterization has revealed that there are striking similarities between human and barramundi IGF-I in their biological and receptor interactions. Interestingly, however, bIGF-I appears to have a greater affinity than hIGF-I for binding to ovine placental membranes. Chemical cross-linking studies with radiolabelled IGFs have previously determined that these membranes are enriched in
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type-2 IGF receptors ŽRead et al., 1986., hence, this result suggests that bIGF-I is more effective than hIGF-I at binding to this receptor. This finding was particularly intriguing since there is no evidence for the type-2 IGF receptor in non-mammalian species and suggested that motifs similar to those required for binding to the mammalian type-2 IGF receptor may be present in fish IGF-Is. Indeed, amino acid sequence comparison of the fish IGF-I sequences identified thus far with those for IGF-II reveal that there are regions of similarity in the two groups of proteins. For example, the fish IGF-I proteins, along with IGF-II, have a greater number of charged residues in the C-domain than are found in the equivalent domain in mammalian IGF-Is. In addition, mammalian IGF-Is have a charged residue at position 50, while the fish IGF-Is and IGF-II have a non-charged residue at the equivalent position. Given that the type-2 IGF receptor appears to have co-evolved with mammals, it is tempting to speculate that we may in fact find that fish IGF-Is have additional or differing functions to their mammalian homologs. A number of studies have previously used human-based immunoassay systems to assess IGF-I levels in fish ŽMoriyama, 1995; Perez-Sanchez et al., 1995.. The data for the radioimmunoassay reported here suggest that this heterologous approach may result in a very large underestimation of the absolute levels of IGF-I in fish. This situation has also been reported by Moriyama et al. Ž1994. who found that use of a homologous immunoassay to measure IGF-I concentrations in salmon indicated that previous studies have underestimated piscine IGF-I concentrations by up to 100-fold. Furthermore, this has also found to be the case in chickens, as the use of a homologous chicken immunoassay demonstrated that circulating and tissue IGF-I concentrations are higher than previously reported using heterologous assays ŽUpton et al., 1992; McMurtry et al., 1994.. In vivo investigation of the clearance of labelled human and barramundi IGF-I in juvenile barramundi has also revealed the importance of using homologous reagents to investigate the IGF system. Thus, radiolabelled hIGF-I was found to be cleared from the circulation of juvenile barramundi more rapidly than the radiolabelled barramundi protein. Moreover, this result can be attributed to differences in affinities of the human and fish proteins for the IGF-binding proteins present in barramundi serum. Taken together, these results represent the first in vivo evidence that there are indeed functional differences between fish and human IGF-Is. Development of a homologous IGF-I immunoassay using an antibody raised against barramundi IGF-I, along with scaled-up production of the protein, will facilitate future in vitro and in vivo investigations of the IGF system in barramundi. If IGFs are involved in as many varied and important functions in fish as is found in mammals, then the benefits of these studies to aquaculture could be substantial.
References Ballard, F.J., Read, L.C., Francis, G.L., Bagley, C.J., Wallace, J.C., 1986. Binding properties and biological potencies of insulin-like growth factors in L6 myoblasts. Biochem. J. 233, 223–230. Francis, G.L., Upton, F.M., Ballard, F.J., McNeil, K.A., Wallace, J.C., 1988. Insulin-like growth factors 1 and
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2 in bovine colostrum: sequences and biological activities compared with those of a potent truncated form. Biochem. J. 251, 95–103. McCormick, S.D., Kelley, K.N., Young, G., Nishioka, R.S., Bern, H.A., 1991. Stimulation of coho salmon growth by insulin-like growth factor I. Gen. Comp. Endocrinol. 86, 398–406. McMurtry, J.P., Francis, G.L., Upton, F.Z., Rosselot, G., Brocht, D.M., 1994. Developmental changes in chicken and turkey insulin-like growth factor-I ŽIGF-I. studied with a homologous radioimmunoassay for chicken IGF-I. J. Endocrinol. 142, 225–234. McMurtry, J.P., Francis, G.L., Upton, Z., Walton, P.E., Rosselot, G., Caperna, T.J., Brocht, D.M., 1996. Plasma clearance and tissue distribution of labelled chicken and human IGF-I and IGF-II in the chicken. J. Endocrinol. 150, 149–160. Moriyama, S., 1995. Increased plasma insulin-like growth factor-I ŽIGF-I. following oral and intraperitoneal administration of growth hormone to rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 99, 221–229. Moriyama, S.D., Duguay, S.J., Conlon, J.M., Duan, C., Dickoff, W.W., Plisetskaya, E.M., 1993. Recombinant coho salmon insulin-like growth factor-I expression in Escherichia coli, purification and characterisation. Eur. J. Biochem. 218, 205–211. Moriyama, S., Swanson, P., Nishii, M., Takahashi, A., Kawauchi, H., Dickoff, W.W., Plisetskaya, E.M., 1994. Development of a homologous radioimmunoassay for coho salmon insulin-like growth factor-I. Gen. Comp. Endocrinol. 96, 149–161. Moriyama, S.D., Dickoff, W.W., Plisetskaya, E.M., 1995. Isolation and characterisation of insulin-like growth factor-I from rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 99, 221–229. Perez-Sanchez, J., Marti-Palanca, M., Kaushik, S.J., 1995. Ration size and protein intake affect circulating growth hormone concentration, hepatic growth hormone binding and plasma insulin-like growth factor-I immunoreactivity in a marine teleost, the gilthead sea bream Ž Sparus aurata.. J. Nutr. 125, 546–552. Read, L.C., Ballard, F.J., Francis, G.L., Baxter, R.C., Bagley, C.J., Wallace, J.C., 1986. Comparative binding of bovine, human and rat insulin-like growth factors to membrane receptors and to antibodies against human insulin-like growth factor-I. Biochem. J. 233, 215–221. Ross, M., Francis, G.L., Szabo, L., Wallace, J.C., Ballard, F.J., 1989. Insulin-like growth factor ŽIGF.-binding proteins inhibit the biological activities of IGF-I and IGF- 2 but not des-Ž1–3.-IGF-I. Biochem. J. 258, 267–277. Upton, F.Z., Francis, G.L., Ross, M., Wallace, J.C., Ballard, F.J., 1992. Production and characterization of recombinant chicken insulin-like growth factor-I from Escherichia coli. J. Mol. Endocrinol. 9, 83–92. Upton, Z., Webb, H., Tomas, F.M., Ballard, F.J., Francis, G.L., 1996. Characterization of serum-derived and recombinant rat IGF-I and their use for measuring true concentrations of IGF-I in rat plasma. J. Endocrinol. 149, 379–387.
In vitro characterization and in vivo clearance of recombinant barramundi žLates calcarifer / IGF-I Brian G. Degger a,b, Neil Richardson b, Chris Collet b, F. John Ballard a , Zee Upton a,) a
CooperatiÕe Research Centre for Tissue Growth and Repair, School of Biological Sciences, GPO Box 2100, Adelaide, SA 5001, Australia b School of Life Sciences, Queensland UniÕersity of Technology, Brisbane, QLD, Australia Accepted 1 October 1998
Abstract Little is known about fish insulin-like growth factors ŽIGFs. as only small amounts have been isolated from native sources or indeed produced recombinantly. This report describes the production of milligram quantities of recombinant barramundi IGF-I ŽbIGF-I. and its subsequent characterization. Recombinant bIGF-I was produced in Escherichia coli using a gene fusion system similar to that previously described for the production of other non-mammalian IGFs. Recombinant bIGF-I was similar to human IGF-I ŽhIGF-I. in stimulating protein synthesis and in competing for binding of labelled hIGF-I to IGF receptors whether tested in rat myoblasts or in salmon embryo fibroblasts. However, recombinant bIGF-I differed from its human counterpart in its affinity for a polyclonal antibody raised against hIGF-I, with at least 200-fold more bIGF-I required to obtain 50% displacement of labelled hIGF-I from the antibody. Hence, the recombinant protein will be essential for developing a specific homologous immunoassay for measuring IGF-I concentrations in barramundi during growth and development. In addition, studies investigating the clearance of labelled bIGF-I and hIGF-I in vivo reveal that the human protein is cleared from the circulation of juvenile barramundi almost twice as fast as the barramundi protein, thus providing the first in vivo evidence that there are functional differences between fish and human IGF-Is. Neutral gel chromatography of serum from the clearance study suggest that this is due to differences in the affinities of the labelled human and fish IGF-I for the IGFBPs present in barramundi. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Barramundi; Lates calcarifer; IGF-I
)
Corresponding author. Tel.: q61-8-8201-5248; Fax: q61-8-8201-3015; E-mail: [email protected] 0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 0 7 5 - 7
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1. Introduction Compared to the situation in mammals, little is known about fish insulin-like growth factors ŽIGFs. and their role in growth and development as only small amounts have been available for such experiments ŽMoriyama et al., 1993, 1995.. However, there is growing evidence from heterologous studies to suggest that IGF-I is also important in fish. Indeed, a number of in vivo studies have shown that elements of the growth hormone–IGF axis are found in fish and that the growth-promoting actions of IGF-I observed in mammals are conserved in fish ŽMcCormick et al., 1991; Moriyama, 1995.. In an attempt to determine whether similar results would be obtained if these studies were undertaken using homologous reagents, recombinant barramundi Ž Lates calcarifer . IGF-I ŽbIGF-I. has been produced using procedures similar to those previously described for the production of milligram quantities of other IGFs ŽUpton et al., 1992, 1996.. While DNA sequences have been reported for a number of fish IGF-Is, production of barramundi IGF-I was pursued due to the importance of this species in aquaculture in the tropical and sub-tropical regions of the Indo-Pacific and Australia. Thus, the aim of the present study was to produce recombinant bIGF-I in quantities to facilitate not only homologous in vitro, but also in vivo investigations.
2. Materials and methods 2.1. Generation of expression construct and fermentation Using site-directed mutagenesis ŽMutagene Kit, BioRad, South Richmond, CA, USA. and a synthetic gene for chicken IGF-I as the starting template, a construct for expression of recombinant barramundi IGF-I ŽbIGF-I. was engineered. The DNA sequence information for the protein was generously provided by Dr. A. Kinhult ŽSchool of Life Sciences, Queensland University of Technology, Brisbane, QLD, Australia.. The engineered construct features codons selected for maximal expression in Escherichia coli. In addition, the expression vector was designed such that when E. coli JM101 Ž lacI q . cells transformed with the construct are fermented, the IGF is expressed as a fusion protein in inclusion bodies ŽUpton et al., 1992.. Fermentation and the subsequent homogenization and centrifugation of the broth to collect the inclusion bodies were performed as described in Upton et al. Ž1992, 1996.. 2.2. Purification of barramundi IGF-I from inclusion bodies Inclusion bodies containing the bIGF-I fusion protein were processed as described by Upton et al. Ž1996. with the exceptions that the fusion protein was refolded in 4 M urea, while the release of the bIGF-I from the fusion partner with hydroxylamine was performed in the presence of 2.5 M urea. Methods for quantitation and chemical analysis of the recombinant protein are also reported in Upton et al. Ž1996..
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2.3. In Õitro characterization The functional properties of recombinant barramundi IGF-I were compared to human IGF-I ŽhIGF-I; GroPep, Adelaide, SA, Australia. in a number of in vitro characterization systems. Stimulation of protein synthesis in L6 rat myoblasts was performed according to Ballard et al. Ž1986.. Assessment of binding to type-1 IGF receptors in cultured cells was performed according to Ross et al. Ž1989., while binding to the type-2 IGF receptors in ovine placental membranes was measured as described by Read et al. Ž1986.. This same publication describes the method used for comparing the cross-reactivities of barramundi and human IGF-I in a radioimmunoassay using a polyclonal antibody prepared against hIGF-I ŽGroPep, Adelaide, SA, Australia.. Radiolabels used in the in vitro characterization assays and in the in vivo clearance study described below were prepared using the chloramine-T procedure ŽFrancis et al., 1988. and were used as indicated in the figures. 2.4. In ÕiÕo clearance of labelled IGF-I in fish Clearance of labelled human and barramundi IGF-I from the circulation of juvenile barramundi Ž90–150 g. was evaluated by administering a bolus injection of the radiolabel intraperitoneally at t s 0 Ž1500 cpmrg body weight. and then sacrificing five fish at each time point Ž1, 2, 4, 6, 10 and 24 h., with the collection of individual serum samples. Determination of TCA-precipitable radioactivity and neutral gel exclusion chromatography of serum samples were performed as described in McMurtry et al. Ž1996..
3. Results 3.1. Fermentation and downstream processing A 10-l fermentation of E. coli transformed with the construct for expression of the bIGF-I fusion protein yielded 55 g of inclusion bodies. Twenty-five grams of the inclusion bodies were processed, resulting in a yield of 20 mg of recombinant bIGF-I. The growth factor recovered from the final step of the downstream processing eluted as a single peak on an analytical high-performance liquid chromatography ŽHPLC. and migrated as a single band with the expected size of approximately 7.3 kDa on a high density sodium dodecyl sulfate ŽSDS.-polyacrylamide gel run under reducing conditions ŽFig. 1.. N-terminal peptide sequencing indicated that the protein had the correct N-terminal sequence and was at least 94% pure based on the average recovery of the major PTH-amino acid from the first five cycles of the Edman degradation. Mass spectrometry indicated that the majority of the protein had a mass 15 Da higher than expected, which, we have now established, is due to the conservative substitution of Ala for Gly 19 . This substitution is the result of a single nucleotide change in the transformed E. coli clone that was fermented.
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Fig. 1. Ža. Analysis of pure recombinant bIGF-I Žlane 2. electrophoresed on a high-density SDS-polyacrylamide gel under reducing conditions. Arrows indicate the size of the molecular mass markers Žlane 1.. Žb. Analysis of pure recombinant bIGF-I on a microbore C4 reverse-phase HPLC column. Elution of protein, monitored as absorbance at 215 nm, was achieved by a gradient of acetonitrile Ždotted line. in 0.1% Žvrv. trifluoroacetic acid.
3.2. In Õitro characterization of bIGF-I The functional properties of recombinant bIGF-I were compared with hIGF-I in a number of in vitro characterization systems. Thus, the abilities of the two peptides to compete for binding of radiolabelled IGF-I to type-1 IGF receptors in CHSE-214 salmon embryo fibroblasts were similar, with half-maximal effects observed at 14.2 " 3.5 ngr0.5 ml and 15.0 " 2.3 ngr0.5 ml for bIGF-I and hIGF-I, respectively. Likewise, the two proteins competed for binding to the type-1 receptors on L6 rat myoblasts to a
Fig. 2. Effects of IGF-Is on binding of labelled hIGF-I to Ža. L6 rat myoblasts and Žb. CHSE-214 salmon embryo fibroblasts. The proteins tested were hIGF-I Ž'. and bIGF-I ŽI.. Values are the means of triplicate determinations at each peptide concentration. ŽSEM values are indicated by vertical bars where they are larger than the symbols..
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Fig. 3. Effects of IGFs on binding of labelled hIGF-II to bovine placental membranes. The proteins tested were hIGF-I ŽB., bIGF-I ŽI. and hIGF-II Žv .. Values are the means of triplicate determinations at each peptide concentration. ŽSEM values are indicated by vertical bars where they are larger than the symbols..
similar extent with half-maximal competition at 2.3 " 0.3 ngr0.5 ml for bIGF-I and 2.0 " 0.2 ngr0.5 ml for hIGF-I ŽFig. 2.. The proteins were also essentially equipotent in their abilities to stimulate protein synthesis in rat myoblasts Žresults not shown.. The human and barramundi proteins differed, however, in their abilities to compete for binding of labelled hIGF-II to ovine placental membranes. While half-maximal effects were not determined for this assay, bIGF-I clearly showed greater ability to compete for binding of the radiolabel than did hIGF-I ŽFig. 3..
Fig. 4. Radioimmunoassay of IGFs using a polyclonal antiserum raised against hIGF-I and hIGF-I as radiolabel. The proteins tested were hIGF-I ŽB. and bIGF-I ŽI.. Values are the means of triplicate determinations at each peptide concentration. ŽSEM values are indicated by vertical bars where they are larger than the symbols..
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Fig. 5. Clearance of labelled IGFs in vivo: percent of intact labelled protein remaining in the circulation over time. The proteins examined were labelled hIGF-I ŽB. and labelled bIGF-I ŽI.. Each point represents the mean value from TCA precipitations on sera from five individual fish at each time point. ŽSEM values are indicated by vertical bars where they are larger than the symbols..
The cross-reactivities of bIGF-I and hIGF-I were compared in an immunoassay using a polyclonal antibody raised against hIGF-I and hIGF-I as the radiolabel. Barramundi IGF-I was 200-fold less effective than human IGF-I at competing for binding to this antibody ŽFig. 4.. 3.3. In ÕiÕo clearance of labelled IGF-I in barramundi Examination of the clearance of radiolabelled hIGF-I and bIGF-I from the circulation of juvenile barramundi revealed interesting differences between the two proteins. Labelled hIGF-I is cleared from the circulation of juvenile barramundi more rapidly than labelled bIGF-I. Indeed, the human protein appears to be removed from the circulation almost twice as fast as bIGF-I ŽFig. 5.. Neutral gel chromatography of serum from the fish in this study suggests that the differences in the clearance of hIGF-I and bIGF-I are due to differences in the affinities for the IGF-binding proteins present in barramundi serum Žresults not shown..
4. Discussion In this report, the production of milligram quantities of recombinant bIGF-I from E. coli and the subsequent characterization of this protein compared with its human counterpart are described. In vitro characterization has revealed that there are striking similarities between human and barramundi IGF-I in their biological and receptor interactions. Interestingly, however, bIGF-I appears to have a greater affinity than hIGF-I for binding to ovine placental membranes. Chemical cross-linking studies with radiolabelled IGFs have previously determined that these membranes are enriched in
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type-2 IGF receptors ŽRead et al., 1986., hence, this result suggests that bIGF-I is more effective than hIGF-I at binding to this receptor. This finding was particularly intriguing since there is no evidence for the type-2 IGF receptor in non-mammalian species and suggested that motifs similar to those required for binding to the mammalian type-2 IGF receptor may be present in fish IGF-Is. Indeed, amino acid sequence comparison of the fish IGF-I sequences identified thus far with those for IGF-II reveal that there are regions of similarity in the two groups of proteins. For example, the fish IGF-I proteins, along with IGF-II, have a greater number of charged residues in the C-domain than are found in the equivalent domain in mammalian IGF-Is. In addition, mammalian IGF-Is have a charged residue at position 50, while the fish IGF-Is and IGF-II have a non-charged residue at the equivalent position. Given that the type-2 IGF receptor appears to have co-evolved with mammals, it is tempting to speculate that we may in fact find that fish IGF-Is have additional or differing functions to their mammalian homologs. A number of studies have previously used human-based immunoassay systems to assess IGF-I levels in fish ŽMoriyama, 1995; Perez-Sanchez et al., 1995.. The data for the radioimmunoassay reported here suggest that this heterologous approach may result in a very large underestimation of the absolute levels of IGF-I in fish. This situation has also been reported by Moriyama et al. Ž1994. who found that use of a homologous immunoassay to measure IGF-I concentrations in salmon indicated that previous studies have underestimated piscine IGF-I concentrations by up to 100-fold. Furthermore, this has also found to be the case in chickens, as the use of a homologous chicken immunoassay demonstrated that circulating and tissue IGF-I concentrations are higher than previously reported using heterologous assays ŽUpton et al., 1992; McMurtry et al., 1994.. In vivo investigation of the clearance of labelled human and barramundi IGF-I in juvenile barramundi has also revealed the importance of using homologous reagents to investigate the IGF system. Thus, radiolabelled hIGF-I was found to be cleared from the circulation of juvenile barramundi more rapidly than the radiolabelled barramundi protein. Moreover, this result can be attributed to differences in affinities of the human and fish proteins for the IGF-binding proteins present in barramundi serum. Taken together, these results represent the first in vivo evidence that there are indeed functional differences between fish and human IGF-Is. Development of a homologous IGF-I immunoassay using an antibody raised against barramundi IGF-I, along with scaled-up production of the protein, will facilitate future in vitro and in vivo investigations of the IGF system in barramundi. If IGFs are involved in as many varied and important functions in fish as is found in mammals, then the benefits of these studies to aquaculture could be substantial.
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