Characterization of recombinant human brain-derived neurotrophic factor variants

The purpose of this work was the chemical characterization of variants of t he recombinant human brain derived neurotrophic factor (rHu-BDNF), expresse d in Escherichia coli. This paper also addresses the question of the in vit ro activity of Obese variants. Chemical characterization of the variants em ployed peptide mapping using Glu-C protease and cyanogen bromide digestion on reduced and alkylated variants followed by the analysis of the digested peptides using mass spectrometry and Edman sequencing. The BDNF variants in this work have been designated by the order of their elution as observed f rom the high temperature RPLC assay. It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxid e instead of methionine at positions 31 and 61, respectively. Peak_4, which is chromatographically a single peak, contained three variants. Two of the se variants had norleucine instead of methionine, at positions 61 and 92, r espectively, while the third had methionine sulfoxide instead of methionine at position 92. Peak_5 had norleucine at position 31 instead of methionine . Ail of these variants showed in vitro biological activity consistent with the BDNF standard, suggesting the preservation of the trkB receptor-ligand binding domain of the variants. (C) 1999 Academic Press.