How To Remove His Tag From Recombinant Protein

Purification Of Membrane Proteins

Expression and purification of His-tagged proteins from E. coli

Polyhistidine-tagged membrane proteins can be purified by IMAC using detergent-containing buffers to solubilize the proteins during the chromatographic process., IMAC of membrane proteins has been carried out successfully in a variety of ionic and nonionic detergents. It is difficult to predict which detergent will be most suitable for IMAC in a given membrane protein system. Although caution should be used, the Ni2+âNTA and Co2+âCMA matrices are generally able to tolerate limited amounts of nonionic and ionic detergents. Following IMAC, it is possible to restore the activity of purified polyhistidine-tagged membrane proteins by reconstitution into membrane vesicles.

Design Of Protein Binding Washing And Elution Steps

Binding of the polyhistidine-tagged proteins can be performed using either a column or a batch procedure. Cell lysis should be done in buffered solution adjusted to pH 8.0. When the column procedure is utilized, the resin is packed into a column and the cell lysate is slowly loaded onto the column. The batch procedure involves incubating the affinity matrix resin in the cell lysate solution and then packing the resin into a column. During incubation at 4°, the resin can be suspended in the cell lysate solution by shaking or stirring. Use of the batch procedure often results in more efficient binding of the tagged protein. With either method, the use of the minimum amount of resin needed to bind the tagged protein is recommended. The tagged protein usually has a higher binding affinity than other proteins that bind nonspecifically to the resin. Thus, when the minimum amount of resin is used, the tagged protein will fill most of the available binding sites, reducing the number of nonspecific proteins that bind. Sodium chloride and low levels of imidazole can also be included in the binding buffer to reduce the number of proteins that bind nonspecifically to the resin. Most protease inhibitors, with the exception of metal-chelating agents such as EDTA, can be included in all buffers. There are commercially available protease inhibitor cocktails specifically designed for use in IMAC purifications .

Expression And Extraction Of Recombinant Amelogenin

Wild-type amelogenin was expressed using a pET28 expression vector modified with a HRV 3C protease site in Rosetta DE3 E. coli cells . Once the cells reached an optical density of 0.61.2 recombinant protein expression was induced by adding isopropyl -D-1-thiogalactopyranoside . Induction of expression was confirmed by analytical SDS PAGE. After shaking overnight incubation at 37°C, the cells were harvested by centrifugation and amelogenin-enriched fractions obtained by extraction in acetic acid as previously reported . In brief, 1 g of pelleted cells were washed by resuspending in 30 mL of 150 mM NaCl. After centrifugation the pellet was resuspended in 30 mL 3% acetic acid, sonicated and heated at 75°C for 20 min. Insoluble material was pelleted by centrifugation and the acetic acid extracts were lyophilized. Lyophilized extracts were dissolved in a minimum volume of 125 mM formic acid and desalted against 125 mM formic acid using size exclusion chromatography . The volatile formic acid was then removed by lyophilization.

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When To Use His Tag Protein Purification

His-tagged fusion protein purification can be used as the only purification step for applications that do not require proteins with extremely high purity. When the highest purity is needed, this technique can be used as the first step in a multistep purification procedure. It is also suitable for purifying dual-tagged proteins with a histidine-tag at one end and a different tag at the other end.

The his tag usually does not have to be removed. In some cases where it interferes with the function of the target protein or the target protein needs to be in a native state, recombinant are used to easily remove his tag after a second pass over the resin used to purify the protein of interest.

Cleavage Of Your Tag From Your Protein Of Interest


Some tags infer little risk to protein functionality but others, especially those of large size, can have downstream implications. In these cases, it is often desirable to cleave the tag from your protein of interest after the initial detection. This can be achieved by using site-specific proteases or inteins.

Site-specific proteases should not affect the functionality of your protein, but it is often desirable to remove the protease after cleavage. One solution is to use a protease that is itself fused to an affinity tag. The protease can then be easily removed via affinity chromatography4. Several protease recognition sites exist depending on your specific need, each with distinct advantages and limitations. One point to consider is whether the tag is fused to the N- or C-terminal.

Cleavage of a tag from the N-terminal will leave minimal excess residues on your protein of interest. On the other hand, cleavage of C-terminal tags will result in 46 extra residues being left on your protein of interest. However, in limited circumstances, carboxypeptidases can be used to remove these short C-terminal sequences1. We have outlined the most common protease sites below.


Cleavage site: DDDDK^X

Advantages: Internal recognition site present in the DDDK tag

Limitations: Variable efficacy dependent on the amino acid after the lysine residue, ranging from 61% 88% . Cleavage can occur at non-specific sites.

Factor X

Cleavage site: IGR^X

Advantages: Widely available.


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How To Use Proteases For Good

We all love to purify proteins that have some kind of tag on it . These tags make our lives much simpler. Dont they?

But it may be necessary to remove these tags when they:

  • Alter the conformation of the protein
  • Interfere with the downstream applications of the proteins
  • Prevent the proteins from being crystallized

Luckily, you can use tag-removing proteases to cleave the tags attached to your proteins and get a pure untagged protein.

How Does It Work

With an affinity tag attached, the target protein is specifically and reversibly bound to a chromatographic resin containing a binding substance with affinity to the tag.

Tagged protein purification step-by-step

  • Apply the sample to the column in conditions that favor the tag and ligand binding. Wash any unbound material out of the column.
  • Elute the bound tagged protein. This is typically done using a competitive ligand.
  • The eluted protein is usually at a high concentration. If you need to remove the tag before using the protein, you can perform cleavage using a site-specific protease.
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    How Many Histidines Should I Add For Protein Expression And Production

    The number of histidine residues in a poly-his-tag can affect the binding of the tagged protein to the affnity resin and elution of the protein off the affinity column. Usually, a hexa-his-tag is sufficient for affinity purification and antibody detection. However, sometime, 7-10-histidines are used to increase binding of the his-tagged protein to the affinity column, possibly due to unexposure of one or more histidine residues to the resin.

    Compatibility With Lysis Buffers From Other Vendors

    Quick Tips Ni-NTA Magnetic Beads for His-tag Purification

    This enhanced membrane technology offers the added convenience of compatibility with different lysis buffers. Cell lysates containing his-tagged protein were prepared with xTractor Buffer and two other lysis buffers from different vendors. No deviations were made from the standard his-tagged purification miniprep kit protocol for any of the samples. Eluates show a high level of compatibility across lysis preps.

    Figure 4. Spin column compatibility with various lysis buffers. Cell lysates prepared with different lysis buffers were all treated according to the standard protocol and purified in duplicate.

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    Protein Purification On Hexahistidine Tag Column


    Charge the His6 tag medium with Ni2+ according to the manufacturer instructions and prepare a 2 × 3 cm column.


    Equilibrate the column with buffer A . Apply the cell lysate to the column at a flow rate of 1 ml/min. Wash the column with buffer A untill no protein can be detected in the flowthrough.


    Elute the affinity-bound proteins with a gradient of 0.020.5 M imidazole. Collect the fractions and take aliquots. Check for the presence of Pacman protein by electrophoresis on a SDSpolyacrylamide gel with subsequent staining with Coomassie blue or by Western blotting.

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    On And Off Column Protein Affinity Tag Removal

      Recombinant proteins are usually expressed with an affinity tag that facilitates binding of the protein to an affinity matrix and results in high yield purification of proteins by exploiting avidity between the tag and affinity matrix. Tags are usually small peptides or proteins that facilitate selective binding to an affinity matrix. There are many types of tags available to facilitate protein purification, some common examples are poly-histidine tag, GST tag, FLAG tag. The bound proteins can be eluted using a chemical agent usually known as eluent that has a higher affinity for affinity matrix and results in the release of bound tagged proteins from the affinity matrix by chemically competing against the bound proteins for the binding sites on affinity matrix.

      Once the purification of proteins is complete these tags may become unwanted because they interfere with the biophysical and biochemical attributes of the purified protein. Tags can change protein stability protein conformation inhibition of enzymatic activity alterations in biological function/activity undesired changes in protein structure for structural studies toxicity. For example, one of the most commonly used tag, hexahistidine tag can influence protein folding, stability and thermotolerance. Not all tags behave similarly, some affinity tags such as MBP and GST tags can significantly improve protein solubility and stability.

      On column Tag removal

      Off column Tag removal

      Analytical Sds Page And Western Blotting

      Expression and purification of recombinant proteins in ...

      Analytical SDS PAGE was carried out based upon the method of Laemmli except 20% v/v glycerol was included in the resolving gel. Proteins were separated using a 12% resolving gel at pH 8.8 and a 4% stacking gel at pH 6.8 using Mini-PROTEAN III or Criterion Cell electrophoresis systems . Samples were dissolved in non-reducing SDS PAGE loading buffer . Prior to SDS PAGE samples were heated at 90°C for 1.5 min and run at a constant 200 V until the bromophenol blue reached the bottom of the gel. Loading for each gel was optimized in each case by running trial gels.

      Following electrophoresis, proteins were detected using Coomassie Blue or silver staining or were transferred to nitrocellulose membranes for Western blot analysis. After blocking and washing, the membrane was incubated with primary antibody for 95 min, washed and then incubated for 90 min with secondary antibody . Immunoreactive protein was visualized using DAB with metal enhancer, according to the manufacturer’s instructions.

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      An Advantage Of Affinity

      The ease of overproducing affinity-tagged forms of TEV and rhinovirus 3C proteases in E. coli has inspired the development of a generic protocol for protein purification , that is outlined in . The same affinity tag is attached to both the protein of interest and to an endoprotease. The tagged protein of interest is first purified from a soluble extract by some type of affinity chromatography, depending on the tag that is used. In the second step, the affinity tag is removed from the POI by the correspondingly tagged endoprotease. Finally, the digestion products are subjected to the same form of affinity chromatography a second time, in this case to remove undigested fusion protein substrate, the tagged protease, the cleaved tag, and any endogenous proteins that bound to the affinity resin during the first round of chromatography, leaving only the untagged POI in the unbound effluent. Variations of this strategy have been developed by a number of groups and have found widespread use in the structural genomics community, e.g., , , , .

      A generic strategy for protein purification that utilizes an affinity-tagged endoprotease. See text for discussion.

      Recipe For Tag Removal

      Using tag-removing proteases requires only a few simple steps.

      First, add a protease specific cleavage site between the sequence of the tag and your protein of interest. Note: scan through your protein sequence and confirm that the protease site is not present anywhere else in your protein. If there is another cleavage site, your protein will get chopped at multiple sites. Use free software like PeptideCutter by ExPaSy or PROSPER.

      Second, purify the protein using the affinity tag.

      Third, throw in the tag removal protease and let it do all the work! You will be left with the untagged protein and the excess protease. You can then separate these by size exclusion chromatography or by using the affinity tag now attached to the protease. Clever!

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      Second Round Nickel Column Purification To Remove Uncleaved Recombinants Free His

      A second round of His-tag cleavage was employed to remove cleaved His-tag and other contaminants present in the cleavage reaction mixture including any uncleaved recombinant. Cleavage products in binding buffer were loaded on to the nickel affinity column as described in the Section entitled Affinity Nickel Column Purification of His-Tagged Recombinant Amelogenins Extracted Using Acetic Acid. Unbound proteins were collected in the column flow through. Bound proteins were eluted using a stepped imidazole gradient in order to optimize the separation. Fractions were analyzed by analytical SDS PAGE as described in the Section entitled Analytical SDS PAGE and Western Blotting.

      Can I Cut Off A Skin Tag With Nail Clippers

      Protein Purification: Strep-tag® vs His-tag – benefits and drawbacks of the two systems

      It can be tempting to cut or clip off a skin tag with a sharp blade, nail clippers, or scissors. Only do this with the approval of a healthcare professional, and cleanse the skin and the tool thoroughly to prevent infection. Also, do not cut or clip off medium or large tags doing so can cause bleeding.

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      A Beginners Guide To Tag

      Every biochemist is familiar with proteases. More often than not, proteases cause a lot of anxiety. To this end, a lot of research has been done in developing techniques to prevent the activity of proteases. But some of these proteases can be the good guys too! For example, you can use them to , or as tag-removing proteases in the protein purification process.

      In this article, I will focus on tag-removing proteases.

      How Do You Get Rid Of Thrombin After Cleavage

      Hi, usually after on-column cleavage using Thrombin, it can be completely removed by placing a pre-packed Heparin affinity chromatography column, HiTrap Benzamidine FF column in series after the GSTrap FF column. The removal of thrombin can be verified with an activity assay using the substrate S-2238.

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      Purification Under Native And Denaturing Conditions

      Purification using the polyhistidine tag can be performed under either native or denaturing conditions by IMAC. The use of mild buffer conditions and imidazole as the elutant often yields biologically active purification products. Proteins that remain soluble in the cytoplasm, or that are secreted, usually can be purified using these native conditions. However, purification under native conditions may be hindered if the target protein is insoluble, aggregates in inclusion bodies, or possesses a tertiary structure that occludes the polyhistidine affinity tag. In such cases, proteins can be purified by the use of denaturing conditions such as 6 M guanidinium hydrochloride or 8 M urea during the purification process. Interaction of the resin with the polyhistidine tag does not require a specific conformation of the peptide tag, which makes effective purification with the use of denaturing conditions possible. Purification under denaturing conditions can depress the activity of phosphatases and proteolytic enzymes. The use of urea as a denaturant is often preferable as 6 M guanidinium hydrochloride precipitates in the presence of SDS, interfering with subsequent SDSâPAGE analysis. Proteins purified under denaturing conditions can then be refolded into their active states by dialyzing away the denaturants. In some cases, proteins can be refolded while bound to the resin.

      How Do I Cleave Of The Poly

      When your his

      In some applications, it is desirable to remove the his-tag, for example, for protein crystallization. To allow cleavage of the tag, a protease cleavage site needs to be engineered between the tag and the protein. An EK cleavage site behind the His-tag can allow complete removal of the tag and the cleavage site, leaving no additional amino acids after the specific cleavage of the tag, resulting in a native form of the protein. For more information on the cleavage site and tag removal of EK and HRV-3C, please refer to: , .

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      Affinity Nickel Column Purification Of His

      Lyophilized crude extracts of recombinant amelogenin were dissolved in a minimum volume of nickel column binding buffer , loaded on to a nickel affinity chromatography column up to a maximum protein load of 40 mg and eluted at a flow rate of 4 mL/min. Unbound proteins were collected. Bound recombinant amelogenin was eluted by increasing the imidazole concentration to 200 mM. The recombinant amelogenin was then buffer exchanged into 125 mM formic acid using size exclusion chromatography and the fractions containing recombinant amelogenin collected and lyophilized. Fractions were analyzed by analytical SDS PAGE and anti-amelogenin Western blotting using rabbit IgGs raised against a peptide corresponding to the amelogenin hydrophilic C-terminal as described in the Section entitled Analytical SDS PAGE and Western Blotting.

      Exceptional Performance With A Wide Range Of Experimental Conditions

      Purification of recombinant proteins may involve many factors that could prove problematic for traditional systems, including denaturing conditions and the presence of additives in the samples and/or buffers. His-tagged purification miniprep kits maintain high performance levels in the face of all these potential complications. A variety of additives were tested at various concentrations during the purification of 6xhis-tagged GFPuv . Protein purification was successful under the standard protocol in the presence of ethylenediaminetetraacetic acid , beta-mercaptoethanol , trisphosphine , dithiothreitol , and glycerol. In all cases, the majority of bound protein was recovered in the first elution, with eluates ranging from 75 µg to > 200 µg per column.

      Figure 2. Purification of GFPuv in the presence of a wide range of additives, at varying concentrations. Additives were included in the sample, equilibration, wash, and elution buffers for each experiment. Samples were eluted twice with 300 µl of elution buffer each time.

      Purification of the same his-tagged protein was also compared under native and denaturing conditions. The standard protocol was applied, and the resulting gel imagery shows excellent purification for all samples, regardless of denaturing conditions.

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