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. Author manuscript; available in PMC: 2021 Mar 19.
Published in final edited form as: Methods Mol Biol. 2017;1586:221–230. doi: 10.1007/978-1-4939-6887-9_14

Removal of Affinity Tags with TEV Protease

Sreejith Raran-Kurussi, Scott Cherry, Di Zhang, David S Waugh
PMCID: PMC7974378  NIHMSID: NIHMS1671727  PMID: 28470608

Abstract

Although affinity tags are highly effective tools for the expression and purification of recombinant proteins, they generally need to be removed prior to structural and functional studies. This chapter describes a simple method for overproducing a soluble form of a stable variant of tobacco etch virus (TEV) protease in Escherichia coli and a protocol for purifying it to homogeneity so that it can be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. Further, we cleave a model substrate protein (MBP-NusG) in vitro using the purified TEV protease to illustrate a protease cleavage protocol that can be employed for simple pilot experiments and large-scale protein preparations.

Keywords: Affinity chromatography, Affinity tag, Fusion protein, His-tag, IMAC, Immobilized metal affinity chromatography, Maltose-binding protein, MBP, TEV protease, Tobacco etch virus protease

1. Introduction

The use of fusion technology has become a widespread practice in the production of recombinant proteins for various applications. Although it was originally designed to facilitate the detection and purification of proteins, subsequently it has become clear that some fusion tags offer extra benefits like improving the yield of their fusion partners, protecting them from intracellular proteolysis, enhancing their solubility, and even facilitating their folding [1]. However, all tags, whether large or small, have the potential to interfere with the structure and function of purified proteins [2–5]. For this reason, it is generally advisable to remove the tag(s) at some stage.

The stringent specificity of viral proteases makes them attractive tools for removing affinity tags. The nuclear inclusion protease from tobacco etch virus (TEV) is probably the best-characterized enzyme of this type. TEV protease recognizes the amino acid sequence ENLYFQ/G with high efficiency and cleaves between Q and G. Its stringent sequence specificity, ease of production, and ability to tolerate a variety of residues at the P1′ position of its recognition site have contributed to its popularity as an endoproteolytic reagent [6, 7].

Here, we describe a method for the large-scale production of a highly active and stable variant (L56V, S135G, S219V mutant) of TEV protease in E. coli and its purification to homogeneity. The protease is initially produced as a fusion to the C-terminus of MBP, which causes it to accumulate in a soluble and active form rather than in inclusion bodies. The fusion protein cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV protease catalytic domain with an N-terminal polyhistidine tag. The His7-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. An S219V mutation in the protease reduces its rate of autolysis by approximately 100-fold and also yields an enzyme with greater catalytic efficiency than the wild-type protease [8]. The L56V and S135G mutations enhance the stability and solubility of the pro tease [9]. The presence of a polyhistidine (His7-tag) on the N-terminus of the protease facilitates not only its purification but also its separation from the digestion products of a His-tagged fusion protein in a subtractive IMAC procedure [10]. We also describe a simple and rapid method to test the solubility of proteins after removing their N-terminal fusion tags in a crude cell lysate.

2. Materials

2.1. Overproduction of His7-TEV (L56V, S135G, S219V) Protease in E. coli

  1. A glycerol stock of E. coli BL21(DE3) CodonPlus-RIL cells containing the TEV protease expression vector pDZ2087 (see Note 1).

  2. LB medium and LB agar plates containing 100 μg/mL ampicillin (for pDZ2087 selection) and 30 μg/mL chloramphenicol (for pRIL selection). LB medium: Add 10 g of Bacto tryptone, 5 g of Bacto yeast extract, and 5 g of NaCl to 1 L of H2O and sterilize by autoclaving. For LB agar, also add 12 g of bacto agar before autoclaving. To prepare plates, allow medium to cool until flask or bottle can be held in hands without burning, then add 1 mL of ampicillin stock solution (100 mg/mL in H2O, filter sterilized), mix by gentle swirling, and pour or pipet ca. 30 mL into each sterile petri dish (100 mm dia.).

  3. Isopropyl-thio-β-D-galactopyranoside (IPTG), dioxane-free. Prepare a stock solution of 200 mM in H2O and filter sterilize. Store at −20 °C.

  4. Shaker/incubator.

  5. Sterile baffled-bottom flasks.

  6. A high speed centrifuge (e.g., Sorvall refrigerated centrifuge).

  7. A spectrophotometer and cuvette that can measure absorbance at 600 nm.

2.2. Purification of His7-TEV (L56V, S135G, S219V) Protease

  1. Cell lysis/IMAC equilibration buffer: 50 mM sodium phosphate (pH 8.0), 200 mM NaCl, 25 mM imidazole. Filter through a 0.22 μm polyethersulfone membrane and store at 4 °C.

  2. A mechanical device to disrupt E. coli cells (e.g., a sonicator, French press, or cell homogenizer) (see Note 2).

  3. Polyethersulfone filtration unit (0.22 and 0.45 μm).

  4. A solution of 5% (w/v) polyetheleneimine, pH 8.0. Mix 50 mL of 50% (w/v) polyethylenimine with H2O to a volume of 450 mL. Adjust the pH to 8.0 with concentrated HCl, and let cool to room temperature. Adjust the volume to 500 mL with H2O and check the pH. Adjust if necessary. Filter through a 0.22 μm polyethersulfone filtration unit. The solution is stable for at least 3 years when stored at 4 °C.

  5. A spectrophotometer and cuvette that can measure absorbance at 280 nm.

  6. ÄKTA Explorer chromatography system or the equivalent.

  7. Ni-NTA Superflow resin (Qiagen Incorporated).

  8. Column XK 26/20 (Amersham Biosciences).

  9. IMAC equilibration buffer: 50 mM sodium phosphate (pH 8.0), 200 mM NaCl, 25 mM imidazole. Filter through a 0.22 μm polyethersulfone membrane and store at 4 °C.

  10. IMAC elution buffer: 50 mM sodium phosphate (pH 8.0), 200 mM NaCl, 250 mM imidazole. Filter through a 0.22 μm polyethersulfone membrane and store at 4 °C.

  11. 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 8.0 stock solution.

  12. 1 M stock solution of 1,4-dithio-dl-threitol (DTT). Prepare

  13. mL by mixing 1.55 g of DTT with H2O to a final volume of 10 mL. Place solution on ice. Use immediately or store at −20 °C.

  14. An Amicon Stirred Ultrafiltration Cell concentrator and YM10 ultrafiltration membranes (Millipore Corporation).

  15. A HiPrep 26/60 Sephacryl S-100 HR column (GE Healthcare Life Sciences).

  16. Gel filtration buffer: 25 mM sodium phosphate (pH 7.5), 100 mM NaCl. Filter through a 0.22 μm polyethersulfone membrane and store at 4 °C.

  17. A Dewar flask filled with liquid nitrogen.

3. Methods

3.1. Overproduction of Soluble His7-TEV Protease in E. coli

The induction of pDZ2087 with IPTG produces an MBP fusion protein (Fig. 1) that self-cleaves in vivo to generate a soluble His7-TEV (L56V, S135G, S219V) protease. Virtually all the protease remains soluble after intracellular processing if the temperature is reduced from 37 to 30 °C after the addition of IPTG.

Fig. 1.

Fig. 1

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A schematic map of the modified TEV protease expression vector pDZ2087 that produces a fusion protein product with the configuration MBP-ENLYFQ/G-His7-TEV. Self-cleavage of the MBP fusion protein by TEV protease generates His7-TEV protease in vivo. (The TEV site is underlined and the site of cleavage is marked by a forward slash in the text above)

  1. Inoculate 50–150 mL of LB broth containing 100 μg/mL ampicillin and 30 μg/mL chloramphenicol in a 500 mL baffle-bottom shake flask from a glycerol stock of pDZ2087 transformed E. coli BL21(DE3) CodonPlus-RIL cells. Place in an incubator and shake overnight at 250 rpm and 37 °C.

  2. Add 25 mL of the saturated overnight culture to each 1 L of fresh LB broth containing 100 μg/mL ampicillin, 30 μg/mL chloramphenicol, and 0.2% (w/v) glucose in a 4 L baffle-bottom shake flask. To ensure that there will be an adequate yield of pure protein at the end of the process, we ordinarily grow 4–6 L of cells at a time.

  3. Shake the flasks at 250 rpm and 37 °C until the cells reach mid-log phase (OD600nm ∼ 0.5), approximately 2 h.

  4. Shift the temperature to 30 °C and induce the culture(s) with IPTG at a final concentration of 1 mM (5 mL of 200 mM IPTG stock solution per liter of culture). Continue shaking at 250 rpm for 4–5 h.

  5. Recover the cells by centrifugation at 5000 × g for 10 min at 4 °C, and store at −80 °C. A 6 L preparation typically yields 30–40 g of cell paste.

3.2. His7-TEV Protease Purification

His7-TEV (L56V, S135G, S219V) protease can be purified to homogeneity in two steps: immobilized metal affinity chromatography (IMAC) using Ni-NTA Superflow resin followed by size exclusion chromatography. An example of a purification monitored by SDS-PAGE is shown in Fig. 2 (see Note 3).

Fig. 2.

Fig. 2

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Purification of His7-TEV (L56V, S135G, S219V) protease monitored by SDS-PAGE (NuPage 4–12% gradient MES gel). M molecular weight standards (kDa). Lane 1: total intracellular protein after induction. Lane 2: soluble cell extract. Lane 3: pooled peak fractions after IMAC. Lane 4: pooled peak fractions after gel filtration and concentration

  1. All procedures are performed at 4–8 °C. Thaw the cell paste from 6 L of culture on ice and suspend in ice-cold cell lysis/ IMAC equilibration buffer (10 mL/g cell paste).

  2. Lyse the cell suspension see Note 2) and measure the volume using a graduated cylinder. Add polyethylenimine to a final concentration of 0.1% (1:50 dilution of the 5% stock solution at pH 8.0) and mix gently by inversion. Immediately centrifuge at 15,000 × g for 30 min.

  3. Apply the supernatant to a 25 mL Ni-NTA superflow column equilibrated in cell lysis/IMAC equilibration buffer (see Note 4). Wash the column with equilibration buffer until a stable baseline is reached (approximately seven column volumes) and then elute the bound His7-TEV (L56V, S135G, S219V) with a linear gradient to 100% elution buffer over ten column volumes.

  4. Pool the peak fractions containing the protease and measure the volume. Add EDTA to a final concentration of 2 mM (1:250 dilution of the 0.5 M EDTA, pH 8.0 stock solution), and mix well. Add DTT to a final concentration of 5 mM (1:200 dilution of the 1 M DTT stock solution), and mix well.

  5. Concentrate the sample approximately tenfold using an Amicon stirred ultrafiltration cell fitted with a YM10 membrane. Remove any precipitate by centrifugation at 5000 × g for 10 min. Estimate the concentration of the partially pure protein solution spectrophotometrically at 280 nm using a molar extinction coefficient of 32,290 M−1 cm−1. The desired concentration is between 5 and 10 mg/mL.

  6. Apply 5 mL of the concentrated sample onto a HiPrep 26/60 Sephacryl S-100 HR column equilibrated with gel filtration buffer. The volume of sample loaded should be no more than 2% of the column volume and contain no more than 50 mg of protein.

  7. Pool the peak fractions from the gel filtration column(s) of pure His7-TEV (L56V, S135G, S219V) protease and concentrate to 1–5 mg/mL (see Subheading 3.2, step 5). Filter through a 0.2 μm syringe filter, aliquot and flash freeze with liquid nitrogen. Store at –80 °C. The final yield of the purified TEV protease is approximately 7.0 mg per gram of wet E. coli cell weight (∼250–300 mg from 6 L of cells).

3.3. Cleaving a Fusion Protein Substrate (MBP-NusGHis6) with TEV Protease

The standard reaction buffer for TEV protease is 50 mM Tris–HCl (pH 8.0), 0.5 mM EDTA and 1 mM DTT, but the enzyme has a relatively flat activity profile at pH values between 4 and 9 and will tolerate a range of buffers, including phosphate, MES, and acetate. TEV protease activity is not adversely affected by the addition of glycerol or sorbitol up to at least 40% (w/v). The enzyme is also compatible with some detergents [11]. TEV protease activity is not inhibited by PMSF and AEBSF (1 mM), TLCK (1 mM), Bestatin (1 mg/mL), pepstatin A (1 mM), EDTA (1 mM), E-64 (3 mg/mL), or “complete” protease inhibitor cocktail (Roche).

However, zinc will inhibit the activity of the enzyme at concentrations of 5 mM or greater, and reagents that react with cysteine (e.g., iodoacetamide) are potent inhibitors of TEV protease. The duration of the cleavage reaction is typically overnight. A good rule of thumb is to use 1 OD280 of TEV protease per 100 OD280 of fusion protein for an overnight digest. TEV protease is maximally active at 34 °C [12], but we recommend performing the digest at 4 °C.

  1. 100 μg of a partially pure fusion protein (MBP-NusG-His6) with a canonical TEV protease recognition site (ENLYFQG) in the linker region [10] is incubated overnight at 4–8 °C in 50 μL of standard reaction buffer (see Subheading 3.3) in the absence or presence of 5.0 μg His7-TEV protease. The reaction products are separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue.

  2. 10 μg of pure His7-TEV protease is added to 100 μL of soluble crude extract containing the MBP-NusG-His6 fusion protein and incubated at room temperature for 1 h. The reaction products are separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (see Note 5).

The results of a typical TEV protease digest of a fusion protein substrate (MBP-NusG-His6) are shown in Fig. 3. Panels A and B represent the digestion of a partially purified sample of MBP- NusG-His6 fusion protein and the digestion of the same fusion protein performed in a crude cell lysate, respectively. The MBP-NusG-His6 fusion protein was affinity purified using an IMAC column [7].

Fig. 3.

Fig. 3

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Digestion of a fusion protein substrate by His7-TEV (L56V, S135G, S219V) protease. (a) 100 μg of the pure substrate, a fusion between E. coli maltose-binding protein (MBP) and Aquifex aeolicus NusG with a canonical TEV protease recognition site (ENLYFQG) in the linker region [10] was incubated overnight at 4 °C in 50 μl of standard reaction buffer (see Subheading 3.3) in the absence (Lane 2) or presence (Lane 3) of 5.0 μg His7-TEV protease. The reaction products were separated by SDS-PAGE (NuPage 4–12% gradient MES gel) and visualized by staining with Coomassie Brilliant Blue. Lane 4 contains an equivalent amount of pure His7-TEV protease. Lane 1 is crude MBP-NusG-His6 (soluble protein) before IMAC purification. (b) Lane 1 is MBP-NusG-His6 soluble protein (crude sample); Lane 2 is TEV protease digest of lane 1 sample, soluble protein and Lane 3 contains an equivalent amount of pure His7-TEV protease used in the cleavage reaction (∼ 0.1 mg/mL). M molecular weight standards (kDa)

3.4. Troubleshooting

Some fusion proteins are intrinsically poor substrates for TEV protease. This may be due to steric occlusion when the protease cleavage site is too close to ordered structure in the passenger protein, or when the fusion protein exists in the form of soluble aggregates. Sometimes this problem can be mitigated by using a large amount of TEV protease (we have occasionally used up to 1 OD280 of TEV protease per 5 OD280 of fusion protein) and/or performing the reaction at higher temperature (e.g., room temperature). Failing that, the addition of extra residues between the TEV protease cleavage site and the N-terminus of the target protein is advised. We have used polyglycine, polyhistidine, and a FLAG-tag epitope with good results.

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Footnotes

1.

E. coli BL21(DE3) CodonPlus-RIL cells containing pDZ2087 can be obtained from our laboratory or from the nonprofit distributor of biological reagents AddGene, Inc., Cambridge, MA, USA (http://www.addgene.org) for a nominal shipping and handling fee. The pRIL plasmid is a derivative of the p15A replicon that carries the E. coli argU, ileY, and leuW genes, which encode the cognate tRNAs for AGG/AGA, AUA, and CUA codons, respectively. pRIL is selected for by resistance to chloramphenicol. Due to the presence of several AGG and AGA codons in the TEV protease coding sequence, the presence of pRIL dramatically increases the yield of TEV protease.

2.

We routinely break cells using a APV-1000 homogenizer (Invensys, Roholmsvej, Germany) at 10–11,000 psi for 2–3 rounds. Other homogenization techniques such as French press, sonication, or manual shearing should yield comparable results. Centrifugation of the disrupted cell suspension for at least 30 min at 30,000 × g is recommended. Filtration through a 0.45 μm polyethersulfone (or cellulose acetate) membrane is helpful to remove residual particulates and fines prior to chromatography.

3.

We find it convenient to use precast gels for SDS-PAGE gels (e.g., 1.0 mm × ten well, 10–20% Tris–glycine gradient).

4.

We use an ÄKTA Explorer chromatography system and Ni-NTA Superflow resin. A properly poured 25 mL Ni-NTA Superflow column (in an Amersham Biosciences XK26/20 column) can be run at 4–6 mL/min (backpressure less than 0.4 MPa) and will bind up to 200 mg of His7-TEV (L56V, S135G, S219V) protease. If a chromatography system is not available, the IMAC can be performed using a peristaltic pump or manually by gravity. If the latter is used, Ni-NTA agarose should be substituted for Superflow and the elution performed with step increases of imidazole in 25 mM increments. Binding and elution profiles can be monitored spectrophotometrically at 280 nm and by SDS-PAGE. Care must be taken to properly zero the spectrophotometer because imidazole has significant absorption in the UV range.

5.

Digestion of a fusion protein by adding TEV protease to a crude cell lysate is a useful way to gauge the efficiency of processing and determine whether or not the passenger protein will remain soluble after it is cleaved from the affinity tag before any chromatography steps are performed. Samples of the TEV digest are compared by SDS-PAGE before and after centrifugation.

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