Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization

Abstract

Gilthead seabream (Sparus aurata) insulin-like growth factor-I (gsIGF-I) cDNA coding for the mature protein was cloned in a pGEM-3Z vector, and then transferred into prokaryotic expression vector pET-11a and expressed in Escherichia coli BL21(DE3) cells upon induction with isopropyl thiogalactoside. The expressed protein contained within the inclusion-body pellet was solubilized in 4.5 M urea, refolded for 24 h at pH 11.3 in the presence of catalytic amounts of cysteine and purified to over 98% purity, as a monomeric methionyl-gsIGF-I. Amino acid composition and N-terminal sequence confirmed the identity to be the predicted protein. Binding assays of the 125I-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indicating the existence of one or more IGF-binding proteins. In binding experiments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value of hIGF-I was about fourfold lower than that of gsIGF-I. Recombinant gsIGF-I exhibited mitogenic activity in a mouse mammary gland-derived MME-L1 cell line which was approximately 200-fold lower than that of hIGF-1. Binding experiments to intact MME-L1 cells suggests that this difference most likely results from a correspondingly lower affinity for IGF-I receptor in these cells. In contrast, the activities of gsIGF-I and hIGF-I measured by 35S uptake by gill arches from the goldfish (Carassius auratus) were identical, indicating that the recombinant gsIGF-I is biologically active.

Real-time polymerase chain reaction, in situ hybridization and immunohistochemical localization of insulin-like growth factor-I and myostatin during development of Dicentrarchus labrax (Pisces: Osteichthyes)

“…Moreover, the cellular localization of IGF-I and MSTN proteins has also been studied from hatching to adult stages by immunohistochemistry with polyclonal antisera raised against sea bream IGF-I (Fine et al 1997) and MSTN (Radaelli et al 2003b). …”

“…Antisera For IGF-I, polyclonal antibodies against recombinant sea bream IGF-I (Fine et al 1997) were raised in mouse and used at a dilution of 1:100. The anti-MSTN antibody was produced in mouse against sea bream MSTNa, which is expressed in several tissues of this species (Radaelli et al 2003b).…”

Action of IGF-I on Expression of the Long Form of the Leptin Receptor (ObRb) in the Prepubertal Period and Throughout the Estrous Cycle in the Mature Pig Ovary

“…Its biological activity was equal to that of human GH as documented using FDC-P1 cells stably transfected with the rabbit growth hormon receptor (GHR) [6]. IGF-I was prepared in the lab of Dr J. Byatt (Monsanto, St. Louis, MO, USA), and its biological activity was documented using undifferentiated mammary epithelial cells cultured in collagen, serum-free medium [7], and bovine mammary cell line MME-L1 [8]. Ovine leptin (oLEP) was prepared at the Institute of Biochemistry, Food Science and Nutrition and was the gift of Professor Gertler.…”

Gh and igf-i increase leptin receptor expression in prepubertal pig ovaries: The role of leptin in steroid secretion and cell apoptosis

“…Its biological activity was equal to that of human GH as documented using FDC-P1 cells stable transfected with rabbit GHR (Herman et al, 1999). IGF-I was prepared in the laboratory of Dr. Monsanto, and its biological activity was documented using undifferentiated mammary epithelial cells cultured in collagen, in serum-free medium (Peri et al, 1992) and mouse mammary cell line MME-L1 (Fine et al, 1997). Ovine leptin (oLEP) was prepared at the Institute of Biochemistry, Food Science and Nutrition and was a gift of Professor Gertler.…”