PolyHis tag: how length matters in protein purification

Jan 29 2018 0 Comments Tags: Affinity purification, Co-NTA, His-tagged protein purification, IMAC resins, Ni-NTA, Ni-Penta resins, Protein function, Protein structure, recombinant protein production

His tag length influences yield and purity on IMAC column

          From time to time, we would get questions like: why is 6xHis (6 constitutive histidine residues) used in most recombinant proteins that are constructed for purification by IMAC? how about 4xHis or 10xHis? how does the length of the polyHis tag impact on IMAC purification?

          It is true that the most commonly used polyHis tag consists of 6 constitutive histidine residues. Longer or shorter polyHis tags have been used much infrequently, but they have been proven to be effective in purification of various target proteins. The reason for using 6xHis might be the determined through trial and error, as it is the shortest stretch of polyhistidine peptide that can be purified by IMAC to satisfactory purity in high yield.

          In the earliest works of one-step purification of polyHis tagged target proteins by IMAC, 6xHis was successfully utilized to purify recombinant protein constructs to 95% purity in high yield. Importantly, the 6xHis tag appeared to be non-disruptive of the functions and activities of tagged proteins from various expression hosts [1,2,3].


How length of the polyHis tag influences purification on IMAC

          The length of the polyHis tag can influence purification performance on IMAC column, in terms of both yield and purity.

          Multiplicity of histidine residues on a protein is associated with its affinity to the immobilized metal ions [4]. Two histidine residues are sufficient to bind onto the Ni-IDA column, which has very high binding affinity to the histidine [4]. However, a pair of adjacent histidines binds weakly to the Ni-NTA column and will be washed off the column by washing buffers with low concentrations of imidazole. As the number of histidine residues in the stretch increases, binding to the IMAC column gets stronger. Therefore, it requires higher imidazole amount to elute from the column [4,5,6].

          Given that greater number of histidine residues results in higher binding affinity, by extending the polyhistidine tag, the target fusion protein can bind on the IMAC columns more efficiently and more tightly, even it is expressed at a low level. In practice, long His tag is also advantageous in achieving greater purity, because it allows more stringent washes at high imidazole concentration to remove nonspecifically bound contaminants. These advantages become more prominent in applications using highly selective columns, such as Co-NTA and Ni-Penta columns. 10xHis was first successfully utilized to purify a recombinant neurotensin receptor, which was expressed at very low levels in E.coli. 10xHis was able to achieve 340-fold enrichment of the functional receptor to 72% purity on Cobalt NTA columns [7].


When to consider longer polyHis tag

          Since 6xHis tag has been proven effective in purification of many target proteins, it is advisable to always start with 6xHis when constructing a new protein. For many proteins, purification can be improved to satisfaction simply by tweaking purification conditions. However, when little or no improvement after all optimization efforts have been made, you might come across with the idea of tailoring the fusion protein construct. Here are some situations where longer polyHis tags (ie. 8xHis or 10xHis) might be helpful to improve yield and purity of the target protein if they are used:

Impurity or contaminant proteins are persistently “co-purified” with the target protein.

          Some E.coli strains contain histidine-rich proteins, which may by sticky on the IMAC column. For example, an E.coli protein consisting a domain homologous to FKBPs has been detected as a persistent contaminant on IMAC [8]. In many mammalian systems, there is a higher natural abundance of proteins containing consecutive histidine residues that lead to aspecific binding on IMAC. For example, it was found in the nuclear extracts of undifferentiated HeLa cells that OCT6 can be readily purified on Ni NTA column due to its naturally occurring stretch of 6 histidine near the C-terminus as well as another His rich sequence [9]. In another study, TGF-b1 was found being purified by Ni NTA from HEK293 cell culture media using a standard protocol [10]. Disulfide bond formation between the protein of interest and other proteins can also lead to contamination. Furthermore, nonspecific hydrophobic interactions can cause some copurification with the desired protein. In these cases, more stringent binding and washing conditions may be required to minimize persistent contaminations. By extending the number of histidine residues in the fusion tag, the binding of the fusion construct on IMAC will be increased, allowing the use of higher imidazole concentrations throughout the purification steps.

The target protein does not bind well on highly selective IMAC resins

          Selectivity and affinity of IMAC for the His tag are the two sides of the same coin. Highly selective IMAC resins (such as Co-NTA and Ni-Penta) have low affinity to the polyHis tag. In some cases where the expression of target protein is low, capturing of the protein on highly selective IMAC columns becomes challenging. Increasing the number of histidine in the tag can help improving affinity of the tag to the immobilized metal ions, and therefore is able to increase the yield after purification.


Facts to keep in mind for using long polyHis tag

          We may have to say the impact of polyHis length on the target protein sometimes gets beyond its behavior on the IMAC column. Here are a few of potential issues that we need to consider when try to tailor the length of polyHis tag to achieve better purity and yield:

  1. Longer polyHis chain is more flexible, so it might reach specific sites that are involved in protein functions, inhibiting the protein’s activities. Removing the tag might be necessary for downstream analyses.
  2. The flexible long chain might also induce conformational changes of the fusion protein construct by interacting with nonspecific sites, increasing the oligomeric state of protein. This is particularly the case for membrane proteins [11]. Consequently, the fusion protein forms aggregates, being depleted from the soluble fraction.
  3. The expression level of the fusion protein in the host may also be affected. It was suggested that the length of the polyHis tag has a greater negative impact on the protein expression than the tag position [11].
  4. On another aspect, as the longer polyHis tag the higher affinity to the immobilized metal, higher amount of imidazole will be required to elute the target protein bound on the column. For example, in the purification of the recombinant AqpZ protein fused with 10xHis tag on Co-NTA, 500 mM imidazole was needed to elute the 10xHis tagged AqpZ protein, instead of 100 mM imidazole that was sufficient for elution of the 6xHis tagged version [11]. However, exposure to high imidazole concentration is undesirable for some target proteins, as they are sensitive to imidazole and may become unstable in solution containing high amount of imidazole [12]. It is advisable to keep the lowest amount of imidazole whenever possible throughout the protocol.



  1. Hochuli E, Bannwarth W, Döbeli H, Gentz R and Stüber D. Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Bio/Technology. 1988; 6: 1321–1325.
  2. Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg HG. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A. 1991;88(20):8972-6.
  3. Janknecht R, Nordheim A. Affinity purification of histidine-tagged proteins transiently produced in HeLa cells. Gene. 1992;121(2):321-324.
  4. Hemdan ES1, Zhao YJ, Sulkowski E, Porath J. Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc Natl Acad Sci U S A. 1989;86(6):1811-5.
  5. Bornhorst JA and Falke JJ. Purification of Proteins Using Polyhistidine Affinity Tags. Methods Enzymol. 2000; 326: 245–254.
  6. Mohanty AK, Wiener MC. Membrane protein expression and production: effects of polyhistidine tag length and position. Protein Expr Purif. 2004;33(2):311-25.
  7. Grisshammer R, Tucker J. Quantitative evaluation of neurotensin receptor purification by immobilized metal affinity chromatography. Protein Expr Purif. 1997;11(1):53-60.
  8. Wülfing C, Lombardero J, Plückthun A. An Escherichia coli protein consisting of a domain homologous to FK506-binding proteins (FKBP) and a new metal binding motif. J Biol Chem. 1994 ;269(4):2895-901.
  9. Schmitt J, Hess H, Stunnenberg HG. Affinity purification of histidine-tagged proteins. Mol Biol Rep. 1993;18(3):223-30.
  10. Kaur J, Reinhardt DP. Immobilized metal affinity chromatography co-purifies TGF-β1 with histidine-tagged recombinant extracellular proteins. PLoS One. 2012;7(10):e48629.
  11. Mohanty AK, Wiener MC. Membrane protein expression and production: effects of polyhistidine tag length and position. Protein Expr Purif. 2004;33(2):311-25.
  12. Reinhard L, Mayerhofer H, Geerlof A, Mueller-Dieckmann J and Weissa MS. Optimization of protein buffer cocktails using Thermofluor. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013; 69(Pt 2): 209–214.


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