Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.
A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins.
Chaga
G
G
Clontech Laboratories, 1020 East Meadow Circle, Palo Alto, CA 94303, USA.
Hopp
J
J
Nelson
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry / Q000201:enzymology*; D016202:N-Methylaspartate / Q000031:analogs & derivatives / Q000737:chemistry; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D017421:Sequence Analysis; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D003035:Cobalt; D016202:N-Methylaspartate; D009532:Nickel; D012685:Sepharose; D007770:L-Lactate Dehydrogenase; D015032:Zinc