Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization.
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.
Fine
M
M
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Amuly
R
R
Sandowski
Y
Y
Marchant
T A
TA
Chan
S J
SJ
Gertler
A
A
Funkenstein
B
B
D000818:Animals; D001709:Biotechnology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000302:isolation & purification; D010473:Perciformes; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I