Preparation and Characterization of the Recombinant Selenomethionine Analogue of Insulin-like Growth Factor-I |
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Authors: | Freddy M De Bree Andrzej M Brzozowski Pär Gellerfors Göran Karlsson Harriet Rönnholm Umberto Breme Paolo Caccia Geoffrey Taylor Gaetano Orsini |
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Institution: | aDepartment of Chemistry, University of York, Heslington, York, United Kingdom;bPharmacia & Upjohn, Stockholm, Swedenand;cPharmacia & Upjohn, Nerviano, Italy |
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Abstract: | 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. |
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