Abstract
Five structural analogs of human insulin-like growth factor II (IGF II), [Leu27]IGF II, [Glu27]IGF II, des(62-67)IGF II, des(62-67)[Leu27]IGF II and des(62-67)[Glu27]IGF II were constructed by site-directed mutagenesis and expressed as protein A fusion proteins in E. coli BL21 pLysS cells, cleaved with CNBr and purified by affinity chromatography and HPLC. These mutants were tested for their binding affinities to type 1 and type 2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. [Leu27]IGF II exhibits an affinity to the type 2 IGF receptor close to that of wild-type IGF II, but has lost completely the affinity to the type 1 IGF receptor. The results further suggest that the D domain, which is close to Tyr27, forms part of the binding region for the type 1 IGF receptor.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Base Sequence
-
Binding Sites
-
Carrier Proteins / metabolism
-
Chromatography, Affinity
-
Chromatography, High Pressure Liquid
-
Cyanogen Bromide
-
DNA / biosynthesis
-
Escherichia coli / genetics
-
Gene Expression*
-
Humans
-
Insulin-Like Growth Factor Binding Proteins
-
Insulin-Like Growth Factor II / analogs & derivatives
-
Insulin-Like Growth Factor II / genetics*
-
Insulin-Like Growth Factor II / metabolism
-
Molecular Sequence Data
-
Mutagenesis, Site-Directed*
-
Receptors, Cell Surface / metabolism
-
Receptors, Somatomedin
-
Recombinant Fusion Proteins / biosynthesis
-
Staphylococcal Protein A
Substances
-
Carrier Proteins
-
Insulin-Like Growth Factor Binding Proteins
-
Receptors, Cell Surface
-
Receptors, Somatomedin
-
Recombinant Fusion Proteins
-
Staphylococcal Protein A
-
Insulin-Like Growth Factor II
-
DNA
-
Cyanogen Bromide