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

M. Fine, R. Amuly, Y. Sandowski, T. A. Marchant, S. J. Chan, A. Gertler*, B. Funkenstein

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

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 ~ 200-fold lower than that of hIGF-I. 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 gsTGF-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.

Original languageEnglish
Pages (from-to)139-150
Number of pages12
JournalJournal of Endocrinology
Volume153
Issue number1
DOIs
StatePublished - Apr 1997

Fingerprint

Dive into the research topics of 'Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: Subcloning, expression in Escherichia coli, purification and characterization'. Together they form a unique fingerprint.

Cite this