title: Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules.
abstract: A (poly)histidine tag was fused to either the N- or the C-terminus of L-lactate dehydrogenase (LDH) of Bacillus stearothermophilus to facilitate purification and immobilization of these enzymes. The C-terminally tagged enzyme displayed lower activity compared both to the wild-type and to the N-terminally tagged variant. The reason for this loss of activity was investigated by affinity chromatography of the enzymes on a 5'-AMP-Sepharose resin and by size-exclusion chromatography. The C-terminally tagged enzyme could be separated into an inactive, unbound fraction and an active, bound fraction. Further differences between the C-terminally tagged enzyme and the N-terminally tagged and wild-type LDH were observed on size-exclusion chromatography of the three enzymes. These data suggest that the introduction of a "his-tag" at the C-terminus may induce misfolding of the LDH and serve as a warning that the introduction of a (poly)histidine tag can produce unforseen changes in a protein.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology, and Medicine, London, United Kingdom SW7 2AY.
authors.forename: C M
authors.initials: CM
authors.lastname: Halliwell
authors.forename: G
authors.initials: G
authors.lastname: Morgan
authors.forename: C P
authors.initials: CP
authors.lastname: Ou
authors.forename: A E
authors.initials: AE
authors.lastname: Cass
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D001411:Geobacillus stearothermophilus / Q000737:chemistry / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification; D017510:Protein Folding
chemical_list: D006639:Histidine; D007770:L-Lactate Dehydrogenase