Periplasmic cold expression and one-step purification of human dihydrolipoamide dehydrogenase.
Dihydrolipoamide dehydrogenase (LADH) is a FAD-linked subunit of alpha-ketoglutarate, pyruvate and branched-chain amino acid dehydrogenases and the glycine cleavage system. As an oxidoreductase it transfers electrons from the dihydrolipoic acid prosthetic group to the NAD(+) cofactor via its FAD center. Besides its physiological function it is capable of generating harmful reactive oxygen species (ROS) in pathological settings therefore it is implicated in neurodegeneration, ischemia-reperfusion, cancer and several other disorders. Pathological mutants of the enzyme cause severe, sometimes lethal syndromes like hypotonia, metabolic acidosis or inefficiency in development. Recently it has been revealed that LADH is a moonlighting protease when specific mutations in the dimerization surface destabilize the functional homodimer and expose a serine-protease-like catalytic dyad. As the basis of versatile functions of LADH is far from elucidation, there is a constant need for a pure and functional enzyme product for investigations. Several studies used recombinant human LADH before, however, it was generated by more complicated and/or physiologically less compatible protocols than reported here; most papers on functional and structural studies do not even report detailed protocols and characteristics (most importantly the purity) of their protein products. Here we describe the details of an optimized, easy-to-use periplasmic expression and one-step purification protocol for obtaining a highly pure, active and authentic (tag-cleaved) enzyme with the characterization of the protein product. The purified LADH can be used in biophysical and structural studies while the published protocol is easily convertible to a protein labeling procedure.
Ambrus
Attila
A
Department of Medical Biochemistry, Semmelweis University, Hungarian Academy of Sciences, Szentagothai Janos Knowledge Center, 9 Puskin Street, H-1088 Budapest, Hungary. ambrus@puskin.sote.hu
Torocsik
Beata
B
Adam-Vizi
Vera
V
D002702:Chloramphenicol Resistance; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003080:Cold Temperature; D008058:Dihydrolipoamide Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism; D009842:Oligopeptides; D019897:Periplasm / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013104:Spheroplasts
C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D009842:Oligopeptides; D011994:Recombinant Proteins; D007655:Ketoglutarate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase