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 produced in Escherichia coli after modification of an expression plasmid that coded for a chicken IGF-II fusion protein. The bIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, and refolded. The protein was then released from the fusion protein by cleavage with subtilisin BPN'. Finally the protein was purified to homogeneity with a number of HPLC steps. In vitro analysis of recombinant bIGF-II demonstrated decreased potency in stimulating protein synthesis when compared to human and barramundi IGF-I (bIGF-I). The in vivo distribution of radiolabeled bIGF-II and bIGF-I in the circulation and tissue uptake of radiolabeled bIGF-II was also compared in juvenile barramundi (Lates calcarifer). Analysis of trichloroacetic acid-precipitable radioactivity in sequential samples following bolus injection of radiolabeled IGFs revealed that bIGF-II was degraded faster than bIGF-I. Moreover, neutral gel chromatography of these samples suggested this difference may be due to reduced affinity of bIGF-II, compared to blGF-I, for the IGF-binding proteins (IGFBPs) present in the barramundi circulation. Based on these results, it would appear 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 determine how they influence the biological actions of native IGFs.
Degger
B
B
Cooperative Research Centre for Tissue Growth and Repair, School of Biological Sciences, Flinders University of South Australia, Adelaide. brian@automatic.com.au
Richardson
N
N
Collet
C
C
Upton
Z
Z
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005399:Fishes / Q000235:genetics*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000235:genetics*; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000493:pharmacokinetics; D014018:Tissue Distribution
D007457:Iodine Radioisotopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II