Polyhistidine Tag

To remove the polyhistidine tag, a Dna sequence coding for a PreScission site (LEVLFQGP) should be introduced between a polyhistidine tag and the mature protein sequence, past designing appropriate primers and performing PCR, as described higher up

From: Methods in Enzymology , 2021

Modern Methods in Natural Products Chemistry

C. Kinsland , in Comprehensive Natural Products II, 2010

9.xix.6.2.1 Immobilized metal analogousness chromatography

The His tag 248 is past far the most popular affinity tag for purification of recombinant proteins. Typically, the tag is composed of half dozen–10 consecutive histidines at either terminus of the poly peptide of involvement, often separated past a protease-cleavage site. The presence of a His tag enables the use of IMAC for purification. IMAC is a rapid analogousness method based on binding of the side by side histidines of the His tag to an immobilized divalent metal ion (nickel is the well-nigh frequently used, followed past cobalt, but other divalent metals are besides used). The His tag binds to the metal at neutral to slightly basic pH (pH seven.v–8 is typical) and the protein can be eluted past lowering the pH to 4–5, stripping the metal from the polymeric support with loftier concentrations of ethylenediaminetetraacetic acrid (EDTA) or, almost commonly, by competition with imidazole. The method is quick, inexpensive, and straightforward, which has led to widespread adoption. Co-ordinate to Derewenda ninety% of deposited crystal structures are derived from recombinantly produced proteins, with almost threescore% using a His tag for purification. 274

The His tag is small-scale (6–10 amino acids) and frequently does not require removal earlier use of the recombinant protein. In fact, although many crystallographers cleave the tag before screening for crystallization weather, 274,275 Carson et al. showed that the tags more often than not had no significant event on the structure of the attached poly peptide. 276 Withal, the tag may still modify solubility or aggregation land of the purified protein. An additional benefit of the His tag is that its analogousness for divalent metal cations is non dependent on a particular poly peptide fold or secondary structure; therefore, IMAC tin exist performed nether denaturing conditions (6   mol   l−1 guanidine hydrochloride or 8   mol   fifty−1 urea, for instance). This can be useful when the poly peptide cannot be expressed in a soluble form and refolding is being pursued (see Department ix.19.5.7).

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Guide to Protein Purification, 2nd Edition

Arun Malhotra , in Methods in Enzymology, 2009

3.1 His-tag

The His-tag (as well called 6xHis-tag) is one of the simplest and most widely used purification tags, with 6 or more than sequent histidine residues. These residues readily coordinate with transition element ions such as Ni2+ or Co2+ immobilized on beads or a resin for purification. IMAC is the preferred selection as a first stride during the purification of His-tagged proteins, though small batch reactions or spin columns with IMAC chaplet tin be used for expression tests or pocket-size preparations. Metal ions are immobilized using linkages such as Ni(II)-nitrilotriacetic acid (Ni-NTA) or Co2+-carboxymethyl-aspartate on resins and beads available from many commercial sources. IMAC media typically has loftier bounden capacities (five–40 mg of His-tagged protein/ml of media), is relatively low toll, and can easily exist sanitized. For nickel binding media, the metal ion can often exist stripped (using buffers with EDTA) and recharged for multiple employ cycles. Some cobalt based resins (such every bit Talon, Clontech Inc.) utilize proprietary linkages that are more durable and cannot be recharged; such resins tin be reused 3 and four times, but offer the advantage of being more specific for polyhistidine tags and almost no metallic leakage during protein elution. IMAC tin can also exist used under denaturing conditions, since the His-tag does not need a specific poly peptide conformation for metal binding; indeed, bounden to IMAC resins is stronger under denaturing conditions as the His-tag becomes more exposed.

His-tags bind the immobilized metal via the histidine imidazole band, and tagged protein bound on IMAC media tin can be easily eluted using elution buffers with imidazole (100–250   mChiliad) or low pH (iv.v–half dozen). Since some endogenous proteins also exhibit weak binding to IMAC media (Bolanos-Garcia and Davies, 2006), a low level of imidazole (5–20   grandM) should exist included in the loading and wash buffers to minimize nonspecific binding.

While the mild elution weather for IMAC is 1 of the positive aspects of His-tags, intendance has to be taken to avoid EDTA (or EGTA) in whatever of the buffers. Jail cell extracts loaded on IMAC columns should not contain any EDTA, and only EDTA-gratuitous protease inhibitor cocktails should be used for sample preparation. TRIS salts weakly chelate metal ions equally well, and the use of TRIS buffers should be minimized (50   chiliadM or less). Most IMAC media is also very sensitive to reducing agents such as DTT or DTE, and depression levels of β-mercaptoethanol (<10   mM) should be used instead.

The pocket-sized size of the His-tag minimizes interference with the folding and structure of the target protein, and this tag can exist positioned at either the Due north- or C-final ends (Fig.   16.1A). While the tag can be removed by introducing a protease cleavage site, there are many examples of proteins that take been crystallized with intact His-tags and little or no impact on the structure of the tagged poly peptide (Carson et al., 2007). In some cases, the His-tag can actually assist in crystal formation (east.one thousand., Smits et al., 2008). The His-tag can too be used with commercially available His-tag-specific antibodies for protein detection.

There are several variations on the standard 6xHis sequence used in His-tags (Terpe, 2003). These tags intersperse multiple histidines among other residues to lower the charge or improve stability, such as the HAT-tag and 6xHN tag which also bind meliorate to the Coii+-Talon resin (Clontech Inc.), and the MAT-tag (Sigma-Aldrich Inc.).

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Rational Design of Enzyme-Nanomaterials

S.A. Díaz , ... I.L. Medintz , in Methods in Enzymology, 2016

3.4.1 Formation

The His6-Tag was selected for its ease of ratiometric assembly with QDs. This associates through the formation of a metal circuitous is only stable in aqueous medium and any extreme atmospheric condition (i.e., extreme pH or ionic strengths) should be properly tested to clinch peptide conjugation to the particles. As the conjugation is not a covalent bond, extremely dilute solutions may also affect the stability of the construct, and samples should be kept in the nM range. In this example, we will utilize water-soluble QDs with short zwitterionic surface ligands (meaty ligand four, CL-four, encounter Fig. 1; Susumu et al., 2011) In general, the peptide is able to bind to the QD fifty-fifty in the presence of bulkier surface moieties such as PEGylated ligands (Breger, Delehanty, & Medintz, 2015). The QD was selected to grade a skilful FRET pair with the called organic label on the peptide, in this case a 540   nm emitting QD. In general, utilization of the QD as a donor is preferable to its apply as a FRET acceptor (Algar, Wegner, et al., 2012; Petryayeva, Algar, & Medintz, 2013).

1.

Dissolve the peptide in the desired buffer (1   × PBS   +   10   μM CaCl2, ePBS) maintaining a concentration (1–ten   μThousand) that is approximately an guild of magnitude higher than the QD suspension. If the peptide is not specially hydrophilic, a minor amount (25   μL) of DMSO can be added before the buffer.

2.

Decide the desired peptide/QD ratio (see Note viii).

3.

Determine the precise QD concentration utilizing an absorbance spectra and the known extinction coefficient. In general, a concentration of 100–500   nG is recommended, if need be dilute sample with ePBS.

iv.

Calculate required volumes of peptide and QD solutions to obtain the desired ratio. Add the QD solution to the peptide in an eppendorf tube and allow 30   min for the conjugation to be completed (Fig. eleven).

Effigy 11. Schematic of QD–peptide enzyme activity sensor. Left: Before the presence of enzyme activeness the substrate presents terminal FRET acceptors. Correct: In the presence of enzyme activeness the substrate is broken, the acceptor and so diffuses away from the QD. The extended altitude reduces the FRET efficiency, increasing QD emission, and decreasing the acceptor emission.

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Regulators and Effectors of Minor GTPases

Anne-Marie Marzesco , ... Ahmed Zahraoui , in Methods in Enzymology, 2001

Expression of Human δPDE in Escherichia coli

Big amounts of purified δPDE protein are required for biochemical studies and for analyzing its interaction with Rab13. Different methods have been employed to limited proteins of interest either in bacteria or in insect cells using baculovirus expression vectors. Nosotros use the T7 expression arrangement to produce native δPDE. This system, originally developed by Studier et al., xv allows cloning of target cDNAs at sites where they are selectively transcribed by T7 RNA polymerase in vitro and in E. coli. Transcription is controlled by the strong φ10 promoter of T7 RNA polymerase. The coding region of the poly peptide of interest is cloned downstream of a T7 RNA polymerase promoter and and then introduced into an E. coli strain containing a chromosomal re-create of the T7 RNA polymerase gene under control of the inducible lacUV5 promoter. Expression of T7 RNA polymerase in this leaner is induced by improver of isopropylthio-β-D-galactoside (IPTG). Two oligonucleotides have been used to create past polymerase chain reaction (PCR) an NdeI restriction site that includes the ATG initiator codon of δPDE coding sequence and a BamHow-do-you-do site downstream of δPDE termination codon. The NdeI-BamHello insert containing the consummate δPDE coding sequence is cloned into pET-15b (Novagen Inc., Madison, WI). This vector carries a stretch of six consecutive histidine residues adjacent to the cloning site and allows the expression of an Northward-final histidine-v PDE fusion poly peptide. It also contains a thrombin cleavage site that allows removal of the His tag. Nevertheless the presence of His tag does not appear to bear upon the biological activeness of near target proteins.

The His tag binds to divalent cations immobilized on metallic chelation resin, such as nickel resin Ni-NTA (Qiagen GmbH, Germany) or cobalt resin TALON (Clontech, GmbH, Germany). Nether our purification conditions (come across below) cobalt chaplet give amend results than nickel beads.

1.

The recombinant plasmid pET-15b/δPDE is introduced into E. coli strain DH5α, which is convenient to maintain the plasmid and allows for a high-level plasmid yield.

2.

Identify the correct recombinant colonies by PCR using the advisable primers. Plasmid DNA is prepared from positive colonies and sequenced.

iii.

Transform with the resulting plasmid E. coli strain BL21(DE3)plysS and select for ampicillin (50 μg/ml) and chloramphenicol (12.five μg/ml)-resistant transformants. This strain is a lysogen bearing the IPTG-inducible T7 RNA polymerase gene. It likewise bears the plasmid plysS, which carries bacteriophage T7 lysozyme gene and confers resistance to chloramphenicol. T7 lysozyme is a specific inhibitor of T7 RNA polymerase; its presence in a host that carries the inducible cistron for T7 RNA polymerase increases the tolerance for toxic target proteins and allows u.s. to efficiently lyse the cells (due east.g., freezing and thawing). The analysis of transformants for expression of His-δPDE is carried out by a rapid screening.

4.

Choice single colonies of transformants into v ml of prewarmed Luria-Bertani (LB) media containing 100 μg/ml of ampicillin and 12.v μg/ml of chloramphenicol. Grow the cultures overnight at 37°.

5.

Inoculate 5 ml of prewarmed LB media containing 100 μg/ml of ampicillin with 500 μl of the overnight culture, and grow threescore min at 37° with vigorous shaking. Inoculate an actress civilisation to serve as the uninduced control.

half-dozen.

Induce expression 3 hour by adding 0.iv 1000Thou IPTG. The cells are harvested by centrifugation 3 min at 12,000g at iv°, and resuspended in 100 μl of sodium dodecyl sulfate (SDS) sample buffer. Extracts from uninduced and induced culture are boiled 5 min, and 10 μl of each sample is analyzed for expression of His-δPDE poly peptide on a 15% SDS-polyacrylamide gel. Clones expressing the human His–δPDE protein are identified by Coomassie blue staining.

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Constitutive Action in Receptors and Other Proteins, Part A

Ryan T. Nitta , ... Albert J. Wong , in Methods in Enzymology, 2010

2.2 Important buffers

•

Qiagen His Tag Lysis Buffer: 50   mGrand NaHtwoPOfour, 300   chiliadM NaCl, ten   mThousand imidazole, 2   μg/mL aprotinin, 2   μg/mL leupeptin, 100   μg/mL PMSF, l   mG β-glycerolphosphate, pH 8.0

•

Qiagen six× His wash buffer: 50   mM NaHtwoPOiv, 300   chiliadYard NaCl, xx   1000M imidazole, pH 8.0

•

Qiagen elution buffer: 50   gM NaH2PO4, 300   one thousandM NaCl, 20   one thousandM imidazole, pH viii.0

•

Kinase buffer: 25   chiliadM HEPES (pH seven.iv), 25   mM MgCl2, 2   one thousandThou dithiothreitol, 0.ane   mM NaVO4, and 25   mThousand β-glycerophosphate

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Reconstituting the Cytoskeleton

Baoyu Chen , ... Michael K. Rosen , in Methods in Enzymology, 2014

6.2 Tev cleavage and final purification of the WRC

The MBP and Hishalf dozen tags are removed using Tev protease cleavage after the amylose bead purification. Add two   thousandM DTT and 200   μL of three   mg/mL-purified Tev protease to the WRC eluted from the amylose beads and incubate at 22   °C for eight   h. Confirm complete cleavage by SDS-PAGE before continuing (Fig. 4.4A).

After Tev treatment, the untagged WRC is purified using the SOURCE 15Q anion-commutation protocol described in Section 5.iv (Fig. four.4B). Puddle fractions containing pure WRC and concentrate the complex using an Amicon Ultra-15 centrifugal filter unit, MWCO xxx   kDa (Millipore) at 2500   g, 4   °C to reduce the sample book to 10   mL (or 1   mL if purifying less than 10   mg of protein). Remove any aggregates by centrifugation. Apply the supernatant to a HiLoad 26/600 Superdex 200 column (or a 24-mL Superdex 200 10/300 GL column if loading less than 10   mg of poly peptide) (GE Healthcare), equilibrated with the WRC-GF buffer (for full general purposes) or the WRC-100KMEI20G buffer (for actin polymerization assays) (Fig. 4.ivC). Measure protein concentration by OD280nm. Concentrate the purified WRC if desired; at least 10   mg/mL is generally achievable without producing protein precipitation. Aliquot, flash freeze, and store the WRC at −   80   °C. WRC can be stored in this style for at least i year. Nosotros routinely use the Arp2/3-mediated pyrene-actin assembly assay to evaluate the quality of the reconstituted WRC (Chen et al., 2010, 2013; Ismail et al., 2009). As shown in Fig. four.4D, the purified WRC should exhibit marginal basal activity and be readily activated by GMPPNP-spring Rac1 (Fig. iv.fourD).

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1000 Protein Coupled Receptors

Yu Qin , ... Yehia Daaka , in Methods in Enzymology, 2013

4 His-Tag Switch Technique

The fundamental difference between the His-tag switch technique and BST is that an irreversible His-tag is jump to a cysteine thiol grouping instead of a biotin tag (Camerini et al., 2007). Free thiol groups in protein samples are first irreversibly blocked with N-ethylmaleimide, and South-nitrosylated cysteine residues are so reduced past ascorbate followed by labeling with His-tagged peptides. His-tag-labeled proteins are purified on nickel columns followed by trypsin digestion for consistent MS analysis with a predicted mass shift of 271.12   Da. Using this method, 28 peptides respective to 19 proteins were identified from the cerebral cortex using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS or nano-liquid chromatography–electrospray ionization (nano-LC-ESI) MS/MS (Camerini et al., 2007). Advantages of using His-tag labeling include the efficient and specific detection of South-nitrosylated proteins by MS analysis and affordable cost.

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Membrane Proteins—Product and Functional Label

Mohana Mahalingam , James D. Fessenden , in Methods in Enzymology, 2015

4.1 His tag cDNA cloning and protein expression

We typically insert x residue histidine tags (His10) into target proteins to deed equally Cy3/5NTA-binding sites. The binding affinity of these reagents is proportional to the length of the histidine tag, with His10 tags having roughly ten-fold higher binding affinity, relative to His6 tags (Fessenden, 2009; Guignet et al., 2004). Tags are inserted into predicted unstructured loops in the target protein determined either from atomic structures or secondary structure predictions (Section ii.1). To monitor Cy3/5NTA binding to the His10 tag, it is necessary to attach a fluorescent FRET donor to the target protein, thereby enabling binding to be measured as loftier energy transfer between this donor and Cy3/5NTA targeted to an side by side Hisx tag.

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Ubiquitin-dependent Protein Deposition

Jason Hon , Ying Lu , in Methods in Enzymology, 2019

3.1 Purification of ubiquitin and substrates

Process:

1.

Poly-histidine tags are genetically inserted into genes encoding ubiquitin and substrates which are subsequently expressed from a T7 promoter in Escherichia coli (BL21(DE3), NEB) at 37°C with 0.v   mThousand IPTG for iii   h. Substrates requiring C-concluding biotinylation are purified using the Impact™ kit (Thermo Fisher Scientific).

2.

Cells are lysed in a standard purification buffer (HB) by sonication and purified poly peptide is isolated using Ni-NTA resin (Bornhurst & Falke, 2000), following manufacturer's instructions.

three.

Proteins eluted from the Ni-NTA resin are further purified with size-exclusion and ion-substitution chromatography.

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Ribonucleases - Part B

Igor Five. Chernukhin , ... Sarah F. Newbury , in Methods in Enzymology, 2001

Poly peptide Purification on Hexahistidine Tag Cavalcade

1.

Accuse the His6 tag medium (Novagen) with Ni2+ according to the manufacturer instructions and ready a 2   ×   three   cm column.

two.

Equilibrate the cavalcade with buffer A [20   mThousand HEPES-KOH (pH   7.0), 3 1000 urea, 0.5 Chiliad NaCl, 10   mThou ii-mercaptoethanol, 10–20   mThousand imidazole (optimum concentration adamant experimentally)]. Utilise the cell lysate to the column at a menstruum rate of 1   ml/min. Launder the column with buffer A untill no protein can be detected in the flowthrough.

3.

Elute the affinity-bound proteins with a gradient of 0.02–0.5 M imidazole. Collect the fractions and take aliquots. Check for the presence of Pacman protein by electrophoresis on a SDS–polyacrylamide gel with subsequent staining with Coomassie blueish or by Western blotting.

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