Protein day

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As protein tag , affinity tag or epitope tag (English tag for 'marking', 'tag' or 'label') are various, mostly short, amino acid sequences that can be used to mark proteins in biochemistry . The marked proteins belong to the fusion proteins .

Applications

A) His tag already present in the vector, B) inserting the DNA sequence of the His tag with the transgene into the vector .
N -terminal Histag primer sequence from the start codon (left), C -terminal His-tag primer sequence up to the stop codon (right).

The various markings are differently well suited for different purposes. In the course of protein design, they are used in the production of recombinant proteins with an expression vector , or their purification and detection via affinity chromatography , pulldown assays , Western blot , immunohistochemistry , fluorescence microscopy or live imaging (see Modern examination methods in the Cell biology ).

To generate a protein tag, the coding DNA sequence of the protein tag is inserted into the coding DNA sequence of the fusion protein behind the start codon or in front of the stop codon while maintaining the reading frame . This creates an N -terminal or a C -terminal protein tag on the protein during translation .

Occasionally the protein tag needs to be removed from the protein after cleaning, e.g. B. can be achieved by a protease interface or an inducible intein . The proteolysis -based approach uses proteases with a longer recognition sequence that only cut at the interface of the protein tag, e.g. B. the TEV protease, thrombin , factor Xa or enteropeptidase . Inteins can be triggered by thiols or by lowering the pH value .

All protein tags allow one of the following methods:

  • Immunoaffinity chromatographic purification (with the appropriate antibodies)
  • Antibody-based detection (e.g. by Western blot, immunohistochemistry or immunofluorescence)

Some tags have other functions besides binding an antibody or immunoconjugate , such as:

Protein tags

history

The first protein tag was published in 1984 by S. Munro and Pelham HR and consisted of a part of the neuropeptide substance P . The next protein tags in 1988 were the MBP-Tag, the HA-Tag, the His-Tag and the FLAG-Tag .

See also

Individual evidence

  1. G. Rigaut, et al .: A generic protein purification method for protein complex characterization and proteome exploration . In: Nature Biotechnology . 17, No. 10, 1999, pp. 1030-1032. doi : 10.1038 / 13732 . PMID 10504710 .
  2. S. Chong, FB Mersha, DG Comb, ME Scott, D. Landry, LM Vence, FB Perler, J. Benner, RB Kucera, CA Hirvonen, JJ Pelletier, H. Paulus, MQ Xu: Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. In: Gene (1997), Vol. 192 (2), pp. 271-281. PMID 9224900 .
  3. ^ S. Chong, GE Montello, A. Zhang, EJ Cantor, W. Liao, MQ Xu, J. Benner: Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. In: Nucleic Acids Res. (1998), Vol. 26 (22), pp. 5109-15. PMID 9801307 ; PMC 147948 (free full text).
  4. DW Wood, W. Wu, G. Belfort, V. Derbyshire, M. Belfort: A genetic system yields self-cleaving inteins for bioseparations. In: Nat Biotechnol. (1999), Vol. 17 (9), pp. 889-892. PMID 10471931 .
  5. a b L. Xing, W. Wu, B. Zhou, Z. Lin: Streamlined protein expression and purification using cleavable self-aggregating tags. In: Microb Cell Fact. (2011), Vol. 10, p. 42. PMID 21631955 ; PMC 3124420 (free full text).
  6. IS Carrico, BL Carlson, Carolyn Bertozzi : Introducing genetically encoded aldehyde into protein. In: Nature chemical biology. Volume 3, Number 6, June 2007, pp. 321-322, doi : 10.1038 / nchembio878 . PMID 17450134 .
  7. Hansjörg Götzke, Markus Kilisch, Markel Martínez-Carranza, Shama Sograte-Idrissi, Abirami Rajavel: The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications . In: Nature Communications . tape 10 , no. 1 , September 27, 2019, ISSN  2041-1723 , p. 1–12 , doi : 10.1038 / s41467-019-12301-7 , PMID 31562305 , PMC 6764986 (free full text) - ( nature.com [accessed May 6, 2020]).
  8. BA Fong, DW Wood: Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation. In: Microb Cell Fact. (2010), Vol. 9, p. 77. PMID 20959011 ; PMC 2978133 (free full text).
  9. ^ A b Thomas P. Hopp, Kathryn S. Prickett, Virginia L. Price, Randell T. Libby, Carl J. March, Douglas Pat Cerretti, David L. Urdal, Paul J. Conlon: A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. In: Bio / Technology. 6, 1988, p. 1204, doi : 10.1038 / nbt1088-1204 .
  10. ^ BA Griffin, SR Adams, RY Tsien : Specific covalent labeling of recombinant protein molecules inside live cells. In: Science. Volume 281, Number 5374, July 1998, pp. 269-272, PMID 9657724 .
  11. ^ DB Smith, KS Johnson: Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. In: Genes. Volume 67, Number 1, July 1988, pp. 31-40, PMID 3047011 .
  12. a b J. Field, J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, IA Wilson, RA Lerner, M. Wigler: Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. In: Molecular and cellular biology. Volume 8, Number 5, May 1988, pp. 2159-2165, PMID 2455217 , PMC 363397 (free full text).
  13. a b 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 .
  14. B. Zakeri, M. Howarth: Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting. In: Journal of the American Chemical Society. Volume 132, Number 13, April 2010, pp. 4526-4527, doi : 10.1021 / ja910795a . PMID 20235501 .
  15. a b H. Bedouelle, P. Duplay: Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space. In: European Journal of Biochemistry . Volume 171, Number 3, February 1988, pp. 541-549, PMID 3278900 .
  16. PW Ho, ZH Tse, HF Liu, S. Lu, JW Ho, MH Kung, DB Ramsden, SL Ho: Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT) expression in an ELISA-based system. In: PloS one. Volume 8, number 9, 2013, p. E74065, doi : 10.1371 / journal.pone.0074065 , PMID 24040167 , PMC 3765251 (free full text).
  17. ^ AD Keefe, DS Wilson, B. Seelig, JW Szostak : One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. In: Protein expression and purification. Volume 23, Number 3, December 2001, pp. 440-446, doi : 10.1006 / prep.2001.1515 , PMID 11722181 .
  18. G. Veggiani, T. Nakamura, MD Brenner, RV Gayet, J. Yan, CV Robinson, M. Howarth: Programmable polyproteams built using twin peptide superglues. In: Proceedings of the National Academy of Sciences . Volume 113, number 5, February 2016, pp. 1202–1207, doi : 10.1073 / pnas.1519214113 , PMID 26787909 , PMC 4747704 (free full text).
  19. B. Zakeri, JO Fierer, E. Celik, EC Chittock, U. Schwarz-Linek, VT Moy, M. Howarth: Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. In: Proceedings of the National Academy of Sciences . Volume 109, number 12, March 2012, pp. E690-E697, doi : 10.1073 / pnas.1115485109 . PMID 22366317 . PMC 3311370 (free full text).
  20. ^ TG Schmidt, J. Koepke, R. Frank, A. Skerra: Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. In: Journal of molecular biology. Volume 255, Number 5, February 1996, pp. 753-766, doi : 10.1006 / jmbi.1996.0061 , PMID 8636976 .
  21. ^ S. Munro, HR Pelham: Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70. In: The EMBO journal. Volume 3, Number 13, December 1984, pp. 3087-3093, PMID 6526011 , PMC 557822 (free full text).

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