Polyhistidine tag

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Nickel-nitrilotriacetic acid complex.

A polyhistidine tag (synonymous His-Tag , Hexahistidine-Tag , His 6 -tag) is a protein tag that is used for protein purification and for the detection of labeled proteins .

properties

The amino acid sequence of the poly-histidine tag is a sequence of at least six histidines , the gene sequence N -terminal after the start methionine - codon or C -terminal before the stop codon in the open reading frame of a gene cloned is. This creates a fusion protein with a polyhistidine tag. Occasionally, an interface for a protease or an intein is inserted between the protein and the polyhistidine tag in order to enable the tag to be cleaved after protein purification.

The polyhistidine tag binds with micromolar affinity to divalent nickel or cobalt ions and forms a chelate complex . If the ions are immobilized by binding to a solid-coupled chelator , the proteins can be selectively bound with polyhistidine tag in affinity chromatography (more precisely: in metal chelate affinity chromatography) and by rinsing the column with 20 mM imidazole to remove the unbound proteins be freed. In 1987, Ernst Hochuli coupled the NTA ligand to agarose beads to purify proteins. This agarose is usually used as a column bed, nitrilotriacetic acid agarose (Ni-NTA agarose), more rarely also Ni 2+ iminodiacetic acid agarose (Ni-IDA agarose) or Co 2+ carboxymethylaspartate agarose (Co-CMA agarose) . The bound proteins are eluted with a buffer which contains 75 to 300 mM imidazole, an acidic pH (pH 4 for nickel agaroses and pH 6 for cobalt agaroses) or nickel or cobalt ions. Interfering substances are EDTA , some surfactants and reducing agents (e.g. from the sample buffer ). After elution, the imidazole is usually removed by dialysis .

The peptidyl-prolyl-cis-trans-isomerases of the FKBP type (SlyD, 25kDa) from E. coli also binds to the nickel-based column materials . Therefore, a tandem affinity purification is often used for further purification. There are also SlyD-deficient strains of bacteria. Cobalt CMA agarose binds SlyD more weakly.

By using several polyhistidine tags in series, proteins with increasing numbers can be eluted one after the other with increasing imidazole concentration. This means that several proteins with different numbers of hexahistidines can be purified simultaneously and eluted one after the other with increasing imidazole concentrations.

Applications

Polyhistidine tags are used for the purification of recombinant proteins by affinity chromatography, for pulldown assays and - if anti-polyhistidine tag antibodies are used - for methods with immunolabelling (e.g. ELISA , Western blot , immunofluorescence , immunohistochemistry and flow cytometry ) . There are selectively binding fluorescent dyes . A polyhistidine-tagged protein can be immobilized on nickel or cobalt surfaces or adsorbed in nickel-containing membranes .

Individual evidence

  1. ↑ E. Hochuli, W. Bannwarth, H. Döbeli, R. Gentz, D. Stüber: Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. In: Nature Biotechnology. 6, 1988, p. 1321, doi : 10.1038 / nbt1188-1321 .
  2. ^ KJ Petty: Metal-chelate affinity chromatography. In: Current protocols in neuroscience / editorial board, Jacqueline N. Crawley ... [et al.]. Chapter 5 May 2001, S. Unit 5.10, doi : 10.1002 / 0471142301.ns0510s05 . PMID 18428493 .
  3. ↑ P Hengen: Purification of His-Tag fusion proteins from Escherichia coli . In: Trends in Biochemical Sciences . 20, No. 7, 1995, pp. 285-6. doi : 10.1016 / S0968-0004 (00) 89045-3 . PMID 7667882 .
  4. ↑ E. Hochuli, H. Döbeli, A. Schacher: New metal chelate adsorbent selective for proteins and peptides containing neighboring histidine residues. In: Journal of Chromatography A . Volume 411, number, 1987, pp. 177-184, doi : 10.1016 / S0021-9673 (00) 93969-4 .
  5. ↑ AC Gavin, M. Bösche, R. Krause, P. Grandi, M. Marzioch, A. Bauer, J. Schultz, JM Rick, AM Michon, CM Cruciat, M. Remor, C. Höfert, M. Schelder, M . Brajenovic, H. Ruffner, A. Merino, K. Klein, M. Hudak, D. Dickson, T. Rudi, V. Gnau, A. Bauch, S. Bastuck, B. Huhse, C. Leutwein, MA Heurtier, RR Copley, A. Edelmann, E. Querfurth, V. Rybin, G. Drewes, M. Raida, T. Bouwmeester, P. Bork, B. Seraphin, B. Kuster, G. Neubauer, G. Superti-Furga: Functional organization of the yeast proteome by systematic analysis of protein complexes. In: Nature. Volume 415, Number 6868, January 2002, pp. 141-147, doi : 10.1038 / 415141a . PMID 11805826 .
  6. ↑ Mark Howarth, Daniel JF Chinnapen, Kimberly Gerrow, Pieter C Dorrestein, Melanie R Grandy, Neil L Kelleher, Alaa El-Husseini, Alice Y Ting: A monovalent streptavidin with a single femtomolar biotin binding site . In: Nature Methods . 3, No. 4, 2006, pp. 267-73. doi : 10.1038 / nmeth861 . PMID 16554831 . PMC 2576293 (free full text).
  7. ^ C Zhao, LM Hellman, X Zhan, WS Bowman, SW Whiteheart, MG Fried: Hexahistidine-tag-specific optical probes for analyzes of proteins and their interactions . In: Analytical Biochemistry . 399, No. 2, 2010, pp. 237-45. doi : 10.1016 / year from 2009.12.028 . PMID 20036207 . PMC 2832190 (free full text).
  8. ^ J. Zhu, G. Sun: Facile fabrication of hydrophilic nanofibrous membranes with an immobilized metal-chelate affinity complex for selective protein separation. In: ACS applied materials & interfaces. Volume 6, number 2, January 2014, pp. 925-932, doi : 10.1021 / am4042965 . PMID 24377297 .