Production of recombinant masu salmon insulin-like growth factor binding protein-2b1 and its action on pituitary cells

Abstract

Insulin-like growth factor binding protein (Igfbp)-2b is believed to be a major carrier of circulating Igf-1 in salmonids. We cloned cDNAs of two paralogs of igfbp-2b from the liver of masu salmon and produced recombinant Igfbp-2b1 corresponding to the circulating form using a bacterial expression system. The deduced amino acid sequence of masu salmon igfbp-2b1 had a 75.2 % sequence identity with that of masu salmon igfbp-2b2, and 88.7 % and 96.5 % with those of Atlantic salmon and rainbow trout igfbp-2b1, respectively. The coding region of masu salmon igfbp-2b1 cDNA was subcloned into the pET-16b or pET-32a vector and expressed using either a histidine (His)-tag or a thioredoxin (Trx) and His-tag. Recombinant masu salmon (rs) Igfbp-2b1 with the fusion partner was fractionated in the precipitate, solubilized, and isolated using Ni-affinity chromatography. His.rsIgfbp-2b1 and Trx.His.rsIgfbp-2b1 were treated with Factor Xa and enterokinase K, respectively, to remove the fusion partner; only the digestion with enterokinase was successful. After enzymatic digestion, rsIgfbp-2b1 was purified employing reversed-phase high-performance liquid chromatography. The purified rsIgfbp-2b1 was added to a primary culture of masu salmon pituitary cells with or without human (h) IGF-1 to assess its effect on the release of growth hormone (Gh). Although addition of hIGF-1 alone had no effect on Gh release, co-incubation with varying amounts of rsIgfbp-2b1 increased Gh release in a dose-dependent manner. In addition, rsIgfbp-2b1 in the absence of hIGF-1 showed a positive effect on Gh release from salmon pituitary cells. These results suggest that rsIgfbp-2b1 may either have Igf-1–independent action on Gh release or inhibits the suppressive effect of local pituitary Igf-1 on Gh release.