Production, in vitro characterisation, in vivo clearance, and tissue localisation of recombinant barramundi (Lates calcarifer) insulin-like growth factor II

Recombinant barramundi insulin-like growth-factor-II (bIGF-II) has been pro duced in Escherichia roll after modification of an expression plasmid that coded for a chicken IGF-II fusion protein. The bIGF-II fusion protein, depo sited in bacterial inclusion bodies, was dissolved under reducing condition s, desalted, and refolded. The protein was then released from the fusion pr otein by cleavage with subtilisin BPN '. Finally the protein was purified t o homogeneity with a number of HPLC steps. In vitro analysis of recombinant bIGF-II demonstrated decreased potency in stimulating protein synthesis wh en compared to human and barramundi IGF-I (bIGF-I). The in vivo distributio n of radiolabeled bIGF-II and bIGF-I in the circulation and tissue uptake o f radiolabeled bIGF-II was also compared in juvenile barramundi (Lates calc arifer). Analysis of trichloroacetic acid-precipitable radioactivity in seq uential samples following bolus injection of radiolabeled IGFs revealed tha t bIGF-II was degraded faster than bIGF-I. Moreover, neutral gel chromatogr aphy of these samples suggested this difference may be due to reduced affin ity of bIGF-II, compared to bIGF-I, for the IGF-binding proteins (IGFBPs) p resent in the barramundi circulation. Based on these results, it would appe ar that elements important in the function of IGFs have been well conserved during vertebrate evolution. However, to clearly define the IGF system in fish it will be necessary to characterise the IGFBPs present and to determi ne how they influence the biological actions of native IGFs. (C) 2001 Acade mic Press.