Characterization of recombinant human brain-derived neurotrophic factor variants.
The purpose of this work was the chemical characterization of variants of the recombinant human brain derived neurotrophic factor (rHu-BDNF), expressed in Escherichia coli. This paper also addresses the question of the in vitro activity of these variants. Chemical characterization of the variants employed 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 from the high temperature RPLC assay. It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxide instead of methionine at positions 31 and 61, respectively. Peak 4, which is chromatographically a single peak, contained three variants. Two of these variants had norleucine instead of methionine, at positions 61 and 92, respectively, while the third had methionine sulfoxide instead of methionine at position 92. Peak 5 had norleucine at position 31 instead of methionine. All 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.
Sunasara
K M
KM
Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York, 12180, USA.
Cramer
S M
SM
Hauer
C R
CR
Rupp
R G
RG
Shoup
V A
VA
D000478:Alkylation; D001665:Binding Sites; D019208:Brain-Derived Neurotrophic Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D013058:Mass Spectrometry; D008715:Methionine / Q000031:analogs & derivatives / Q000032:analysis; D008970:Molecular Weight; D009646:Norleucine / Q000032:analysis; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010449:Peptide Mapping; D020813:Receptor, trkB / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000378:metabolism
D019208:Brain-Derived Neurotrophic Factor; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009646:Norleucine; D008715:Methionine; D020813:Receptor, trkB; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide; C013111:methionine sulfoxide