Preparation and Characterization of the Recombinant Selenomethionine Analogue of Insulin-like Growth Factor-I

• Published in: Protein expression and purification 1998-08, Vol.13 (3), p.319-325
• Main Authors: De Bree, Freddy M., Brzozowski, Andrzej M., Gellerfors, Pär, Karlsson, Göran, Rönnholm, Harriet, Breme, Umberto, Caccia, Paolo, Taylor, Geoffrey, Orsini, Gaetano
• Format: Article
• Language: English
• Subjects: Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Insulin-Like Growth Factor I - analogs & derivatives; Insulin-Like Growth Factor I - genetics; Insulin-Like Growth Factor I - metabolism; Peptide Mapping; Radioligand Assay; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Selenomethionine - chemistry; Selenomethionine - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1998.0906

Insulin-like growth factor-I (IGF-I), a single-chain polypeptide consisting of 70 amino acids and 3 disulfide bridges, is a member of a class of growth factors that are involved in many proliferative and metabolic processes. To assist in solving the crystallographic three-dimensional structure, we have expressed a recombinant fusion protein precursor of IGF-I in a methionine auxotrophic strain ofEscherichia coligrown in the presence of selenomethionine. An homogeneous preparation of selenomethionyl-IGF-I was then obtained by chemical cleavage of the fusion protein. The selenomethionine analogue of IGF-I was characterized by electrospray mass spectrometry, peptide mapping, analytical chromatography, and electrophoresis as well as by biological assays. The final preparation of IGF-I was found to incorporate about 90% of selenium and fully retained the functional activity.