Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site

Chant, A.D.B.; Kraemer-Pecore, Christina M.; Watkin, R D; Kneale, Geoff

doi:10.1016/j.pep.2004.10.017

Cited by 124 publications

(81 citation statements)

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“…In many cases they are benign, but they can affect protein behavior in complex ways including altering protein dimerization (43,53), conformation (50), and binding to other proteins (54). In this study, we established that N-terminal His tags dramatically increase EB1 microtubule binding and polymerization promoting activity.…”

Section: Variability In Reported Eb1 Behaviors and The Effect Of Hismentioning

confidence: 82%

Interactions between EB1 and Microtubules

Zhu

Gupta

Slabbekoorn

et al. 2009

Journal of Biological Chemistry

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Plus end tracking proteins (؉TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved ؉TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip. Microtubules (MTs)2 are a major component of the cytoskeleton, the network of proteinacious fibers that endow the cell with structural integrity, motile properties, and internal organization (1-4). MTs play a particularly important role in cell organization: they help to pull the chromosomes apart at mitosis, act as a "railway" for intracellular transport, and define the localization and structure of internal membrane systems (5-11).The cellular functions of MTs are highly dependent on their dynamic nature, which is regulated by a number of microtubule associated proteins (MAPs) (3, 9, 12, 13). The plus end tracking proteins (ϩTIPs) are an unusual group of MAPs that preferentially localize to growing MT plus ends (13)(14)(15). A large number of proteins have now been identified as ϩTIPs, but one of the most important is EB1 (end binding protein-1) (4,13,16,17). EB1 was initially discovered as a binding partner of the adenomatous polyposis coli protein, but it is becoming apparent that EB1 binds to an astonishing array of other proteins including other ϩTIPs (CLIP-170, p150, and CLASPs), molecular motors (Tea2 and kinesin), signal transduction proteins (Rho-GEF2), and cytoskeletal scaffolding proteins (spectraplakins and formins) (4, 13, 15). As might be expected from a protein with so many interactions, EB1 is strikingly well conserved across eukaryotes (18 -21). These observations suggest that EB1 is the structural and evolutionary core of a complex of proteins that regulates the behavior of MT plus ends (4, 13-15). However, its activities and the mechanisms of i...

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Section: Variability In Reported Eb1 Behaviors and The Effect Of Hismentioning

confidence: 82%

Interactions between EB1 and Microtubules

Zhu

Gupta

Slabbekoorn

et al. 2009

Journal of Biological Chemistry

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“…By manipulations of the temperature during expression and dilution refolding, this solubility problem may be able to be solved (Bird et al, 2002). Nevertheless, the length, composition and location of the poly-histidine tag can necessitate additional optimization depending upon the amino acid sequence of the native protein (Chant et al, 2005;Gaberc-Porekar et al, 1999). For instance, the fusion tag can be attached to the N-terminus, C-terminus or both termini of virtually any protein in order to facilitate its purification by IMAC.…”

Section: Challenges In the Use Of Imac And Poly-histidine Tag For Purmentioning

confidence: 99%

“…However, other reports suggest altered biological or physicochemical properties of the histidine tagged proteins as compared to their native counterparts (Arnau et al, 2006;Gaberc-Porekar and Menart, 2005). Introducing a poly-histidine tag can adversely affect the biochemical properties (Horchani et al, 2009), alter the binding characteristics (Gaberc-Porekar et al, 1999), change protein structure conformation (Chant et al, 2005), prompt protein oligomerization (Amor-Mahjoub, 2006), and thus render the purified target unsuitable for use in X-ray crystallography or other characterization studies (Renzi et al, 2006). Consequently, an important part of the design of a fusion tag is the choice of the method for removing the tag after purification.…”

Section: Challenges In the Use Of Imac And Poly-histidine Tag For Purmentioning

confidence: 99%

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions

Kuo

Chase

2011

Biotechnol Lett

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. Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions. Biotechnology Letters, Springer Verlag, 2011, pp.1075-1084.1007/s10529-011-0554-3>. AbstractImmobilized metal affinity chromatography (IMAC) of proteins containing poly-histidine fusion tags is an efficient research tool for purifying recombinant proteins from crude cellular feedstocks at laboratory scale. Nevertheless, to achieve successful purification of large amounts of the target protein for critical therapeutic applications that demand the precise removal of fusion tags, it is important to also take into consideration issues such as protein quality, efficiency, cost effectiveness, and optimal affinity tag choice and design. Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges. In addition, the platform nature of this technology makes it an ideal choice in purifying proteins with unknown properties.Finally, the unique interaction between immobilized metal ions and poly-histidine fusion tag has enabled new developments in the areas of biosensor, immunoassay, and other analytical technologies.

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“…Since fusion tags can modify the behavior of proteins, they offer the possibility for development of standardized protocols for purification, increased solubility, and detection [1][2][3][4][5]. However, fusion tags can also interfere with protein function and with structural studies [6][7][8]. Thus it is often advantageous to remove fusion tags prior to use.…”

Section: Introductionmentioning

confidence: 99%

A combined approach to improving large-scale production of tobacco etch virus protease

Blommel

Fox

2007

Protein Expression and Purification

242

258

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Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in E. coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible.Here we use a quantitative, high throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove 4 of the 5 arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 °C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of ~400 mg/ L of expression culture (~15 mg pure TEV protease per g of E. coli cell paste). Methods for producing glutathione S-transferase tagged TEV with similar yields (~12 mg pure protease fusion per g of E. coli cell paste) are also reported.

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Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site

Cited by 124 publications

References 23 publications

Interactions between EB1 and Microtubules

Interactions between EB1 and Microtubules

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions

A combined approach to improving large-scale production of tobacco etch virus protease

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