Purification and spectroscopic characterization of the recombinant BG21 isoform of murine golli myelin basic protein

✍ Scribed by Vladimir V. Bamm; Mumdooh A.M. Ahmed; Vladimir Ladizhansky; George Harauz

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 691 KB
• Volume: 85
• Category: Article
• ISSN: 0360-4012
• DOI: 10.1002/jnr.21129

✦ Synopsis

Abstract

A recombinant form of the murine Golli‐myelin basic protein (MBP) isoform BG21 (rmBG21) has been expressed in E. coli, and isolated to 96% purity via metal chelation chromatography. Characteristic yields were 6–8 mg protein per liter of culture in either minimal M9 or standard Luria‐Bertani media. Circular dichroism spectroscopy showed that rmBG21 had a large proportion of random coil in aqueous solution, but gained α‐helix in the presence of monosialoganglioside G~M1~ and PI(4)P, as well as in the membrane‐mimetic solvent trifluoroethanol. Bioinformatics analyses of the amino acid sequence of rmBG21 predicted an N‐terminal calmodulin (CaM)‐binding site. It was determined by fluorescence spectroscopy and dynamic light scattering that rmBG21 and CaM interacted weakly in a 1:1 ratio in a Ca^2+^‐dependent manner. Solution NMR spectra of uniformly [^13^C^15^N]‐labeled protein in aqueous buffer were consistent with it being an extended protein; spectral quality was independent of temperature. Thus, like “classic” MBP and the Golli‐MBP isoform J37, rmBG21 is intrinsically disordered, implying multi functionality, and that its conformation depends on its environment and bound ligands. © 2006 Wiley‐Liss, Inc.