sepharose and nickel-nitrilotriacetic-acid

There is a particular need in protein analysis and purification for specific, functional, and generic methods of protein immobilization on solid supports. Here we describe a double-hexahistidine (His6) tag sequence, comprising two hexahistidines separated by an 11-amino acid spacer, which shows at least 1 order of magnitude stronger binding to Ni-NTA-modified surfaces than a conventional single-His6 tag or two single-His6 tags at N- and C-termini. Using, as a model, tagged versions of green fluorescent protein (GFP), stable and tight binding of the double-His6 tag/Ni-NTA interaction was demonstrated by competitive elution from Ni-NTA agarose beads, surface plasmon resonance on a Ni-NTA chip, and ELISA in Ni-NTA microwell plates. Protein purification by Ni-NTA chromatography was improved by a 6-8-fold increase in imidazole concentration required for elution, while the dissociation rate of double-His6 GFP from Ni-NTA chips in SPR (BIAcore) was 10 times slower than for single-His6-tagged proteins. ELISA assays and protein microarrays constructed with double-His6 GFP demonstrated greater detection sensitivity with anti-His antibodies and Ni-NTA conjugates. Moreover, the double-His6 tag could serve simultaneously both for protein immobilization and for detection on surfaces. The double-His6 peptide has the potential to be a universal tag for protein immobilization and detection on arrays and single-step purification of proteins from crude mixtures.

Topics: Binding Sites; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Green Fluorescent Proteins; Histidine; Nitrilotriacetic Acid; Oligopeptides; Organometallic Compounds; Protein Array Analysis; Sensitivity and Specificity; Sepharose; Surface Properties; Time Factors

Antibodies to DNA define an important autospecificity that arises in systemic lupus erythematosus (SLE). To elucidate the molecular features that may explain the pathogenesis of SLE, a heterologous system for expression of cloned V genes is often desirable. Here, a single-chain Fv coding domain was constructed by using the heavy- and light-chain V genes of a high-affinity site-directed mutant of the murine anti-dsDNA autoantibody, 3H9. This scFv was joined in frame to the c-jun leucine zipper for dimerization, and to two affinity tags, domain B of the staphylococcal protein A and a pentahistidine peptide, for purification. Dimerization of the scFv was determined by size-exclusion chromatography. The yields of the scFv following affinity purification on IgG agarose or Ni-NTA agarose were compared, and the activities of the resulting protein fractions were determined. A two-step purification of periplasmic extracts on Ni-NTA agarose and IgG agarose, followed by elution with 3.5 M MgCl(2), yielded scFv with the highest specific activity. The final purified material bound DNA by ELISA, electrophoretic mobility shift assay, and immunofluorescence of fixed Hep-2 cells. Antibodies purified in this fashion should have applications in structure/function studies in which it is essential to generate highly purified antigen-combining sites.

Topics: Affinity Labels; Animals; Antibodies, Antinuclear; Antibody Specificity; Antigen-Antibody Reactions; Autoantibodies; Base Sequence; Chromatography, Affinity; Dimerization; DNA; DNA-Binding Proteins; Durapatite; Escherichia coli; Gene Expression; Humans; Immunoglobulin G; Immunoglobulin Variable Region; Leucine Zippers; Mice; Nickel; Nitrilotriacetic Acid; Organometallic Compounds; Periplasm; Protein Engineering; Recombinant Proteins; Sepharose

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.

Topics: Affinity Labels; Animals; Chelating Agents; DNA, Single-Stranded; Histidine; Immunoglobulin Fab Fragments; Immunoglobulin Variable Region; In Vitro Techniques; Kinetics; Mice; Mutation; Nitrilotriacetic Acid; Oligopeptides; Organometallic Compounds; Sepharose