The use of Ni-nitrilotriacetic acid agarose for estimation of affinities of hexahistidine-tagged Fab to single-stranded DNA.
The complex formed between 32P-labeled (dT)15 and a hexahistidine (6-His)-tagged anti-single-stranded DNA (ssDNA) Fab, DNA-1, was trapped by addition of nickel-chelating nitrilotriacetic acid (Ni-NTA) agarose that led to efficient separation of bound ligand from free. High stability of the immobilized complex (half-life of 4 h) and low nonspecific binding of (32P](dT)15 allowed for a rapid estimation of the dissociation constant (Kd) and was found to be approximately 130 nM. Oligonucleotide bound DNA-1 preimmobilized on Ni-NTA agarose with the same Kd as the Fab/(dT)15 complex formed in solution, indicating that the interaction of the 6-His tag with the resin did not interfere with binding. Addition of unlabeled (dT)15 led to a fast exchange with bound [32P](dT)15. Mutant versions of DNA-1 were also examined and results obtained were in agreement with data from equilibrium gel filtration and fluorescence titration [A. A. Komissarov, M. J. Calcutt, M. T. Marchbank, E. N. Peletskaya, and S. L. Deutscher (1996) J. Biol. Chem. 271, 12241-12246]. These results demonstrate that the Ni-NTA assay is an efficient and accurate method to examine 6-His-tagged protein-nucleic acid complexes. Furthermore, a competition modification of this assay may be used for detection of anti-ssDNA antibodies in serum.
Komissarov
A A
AA
Department of Biochemistry, University of Missouri School of Medicine, Columbia 65212, USA.
Marchbank
M T
MT
Deutscher
S L
SL
D000345:Affinity Labels; D000818:Animals; D002614:Chelating Agents; D004277:DNA, Single-Stranded / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D006639:Histidine; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D009154:Mutation; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives*; D009842:Oligopeptides; D009942:Organometallic Compounds; D012685:Sepharose
D000345:Affinity Labels; D002614:Chelating Agents; D004277:DNA, Single-Stranded; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D009842:Oligopeptides; D009942:Organometallic Compounds; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D012685:Sepharose; D009571:Nitrilotriacetic Acid