Site-specific proteolysis of the Escherichia coli SecA protein in vivo.

A seven-amino-acid cleavage site specific for tobacco etch virus (TEV) protease was introduced into SecA at two separate positions after amino acids 195 and 252. Chromosomal wild-type secA was replaced by these secA constructs. Simultaneous expression of TEV protease led to cleavage of both SecA derivatives. In the functional SecA dimer, proteolysis directly indicated surface exposure of the TEV protease cleavage sites. Cleavage of SecA near residue 195 generated an unstable proteolysis product and a secretion defect, suggesting that this approach could be used to inactivate essential proteins in vivo.     

A new vector for high-throughput,ligation-independent cloning encoding a tobacco etch virus protease cleavage site

To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-independent cloning (LIC) is well suited to robotic cloning and expression, but few LIC vectors are available commercially. We have developed a new LIC vector, pMCSG7, that incorporates the tobacco etch virus (TEV) protease cleavage site into the leader sequence. This protease is highly specific and functions under a wide range of conditions. The new vector incorporates an N-terminal his-tag followed by the TEV protease recognition site and a SspI restriction site used for LIC. The vector functioned as expected, giving high cloning efficiencies and strong expression of proteins. Purification and cleavage of a target protein showed that the his-tag and the TEV cleavage site function properly. The protein was purified and cleaved under different conditions to simulate both plate-based screening methods and large-scale purifications for crystal production. The vector also includes a pair of adjacent, unique restriction sites that will allow insertion of additional modules between the his-tag and the cleavage site of the leader sequence to generate a family of vectors suitable for high-throughput production of proteins.     

The P1' specificity of tobacco etch virus protease

Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the tobacco etch virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.     

Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro

Affinity tags are widely used as vehicles for the production of recombinant proteins. Yet, because of concerns about their potential to interfere with the activity or structure of proteins, it is almost always desirable to remove them from the target protein. The proteases that are most often used to cleave fusion proteins are factor Xa, enterokinase, and thrombin, yet the literature is replete with reports of fusion proteins that were cleaved by these proteases at locations other than the designed site. It is becoming increasingly evident that certain viral proteases have more stringent sequence specificity. These proteases adopt a trypsin-like fold but possess an unconventional catalytic triad in which Cys replaces Ser. The tobacco etch virus (TEV) protease is the best-characterized enzyme of this type. TEV protease cleaves the sequence ENLYFQG/S between QG or QS with high specificity. The tobacco vein mottling virus (TVMV) protease is a close relative of TEV protease with a distinct sequence specificity (ETVRFQG/S). We show that, like TEV protease, TVMV protease can be used to cleave fusion proteins with high specificity in vitro and in vivo. We compared the catalytic activity of the two enzymes as a function of temperature and ionic strength, using an MBP-NusG fusion protein as a model substrate. The behavior of TVMV protease was very similar to that of TEV protease. Its catalytic activity was greatest in the absence of NaCl, but diminished only threefold with increasing salt up to 200 mM. We found that the optimum temperatures of the two enzymes are nearly the same and that they differ only two-fold in catalytic efficiency, both at room temperature and 4 degrees C. Hence, TVMV protease may be a useful alternative to TEV protease when a recombinant protein happens to contain a sequence that is similar to a TEV protease recognition site or for protein expression strategies that involve the use of more than one protease.     

Efficient cleavage of problematic tobacco etch virus (TEV)-protein arginine methyltransferase constructs

Protein arginine methyltransferases (PRMTs) are enzymes that are involved in many biological processes. Several studies have shown that the identity of the N-terminal fusion tag affects the substrate selectivity of PRMTs. Therefore, to accurately study substrate recognition, it is imperative that a tagless PRMT be used. However, cleavage of tagged PRMTs has been problematic. We have developed a successful method by which untagged PRMTs can be made using a tobacco etch virus (TEV) cleavage site at the N-terminal domain. This method may be useful for cleaving other challenging target proteins that have the TEV protease recognition site.     

Controlled intracellular processing of fusion proteins by TEV protease

Here we describe a method for controlled intracellular processing (CIP) of fusion proteins by tobacco etch virus (TEV) protease. A fusion protein containing a TEV protease recognition site is expressed in Escherichia coli cells that also contain a TEV protease expression vector. The fusion protein vector is an IPTG-inducible ColE1-type plasmid, such as a T7 or tac promoter vector. In contrast, the TEV protease is produced by a compatible p15A-type vector that is induced by tetracyclines. Not only is the TEV protease regulated independently of the fusion protein, but its expression is highly repressed in the absence of inducer. Certain fusion partners have been shown to enhance the yield and solubility of their passenger proteins. When CIP is used as a purification step, it is possible to take advantage of these characteristics while both eliminating the need for large amounts of pure protease at a later stage and possibly simplifying the purification process. Additionally, we have observed that in some cases the timing of intracellular proteolysis can affect the solubility of the cleaved passenger protein, allowing it to be directed to either the soluble or the insoluble fraction of the crude cell lysate. This method also makes it possible to quickly gauge the efficiency of proteolysis in vivo, before protein purification has begun and in vitro processing is attempted.     

An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni(2+) affinity chromatography, 65mg of His6-TEV was isolated with purity over 95% from 1L of culture. The enzyme activity of His6-TEV was generally characterized by using GST-EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties.     

Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site

Tobacco etch virus (TEV) protease is a cysteine protease exhibiting stringent sequence specificity. The enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins. Crystal structures of two TEV protease mutants as complexes with a substrate and a product peptide provided the first insight into the mechanism of substrate specificity of this enzyme. We now report a 2.7A crystal structure of a full-length inactive C151A mutant protein crystallised in the absence of peptide. The structure reveals the C terminus of the protease bound to the active site. In addition, we determined dissociation constants of TEV protease substrate and product peptides using isothermal titration calorimetry for various forms of this enzyme. Data suggest that TEV protease could be inhibited by the peptide product of autolysis. Separate modes of recognition for native substrates and the site of TEV protease self-cleavage are proposed.     

A fluorogenic substrate as quantitative in vivo reporter to determine protein expression and folding of tobacco etch virus protease in Escherichia coli

Quantitative and folding reporters are adequate tools to optimize recombinant protein expression in various host organisms, including Escherichia coli. To determine the yield of soluble active protease from the tobacco etch virus (TEV), we developed a single-molecule assay based on the fluorogenic substrate ANA-QS-MCA. This substrate consists of a 10 amino acid peptide (ENLYFQSGTK) containing the proteolytic cleavage sequence of the TEV protease. The peptide works as a linker N-terminally tagged with a fluorescent donor group (7-Methoxycoumarin-4-yl)acetyl (MCA) and C-terminally tagged with the acceptor group 5-Amino-2-nitrobenzoic acid (ANA). Fluorescence can be observed after specific cleavage of the substrate at the Gln-Ser bond by active TEV protease. Purified His-tagged TEV protease was used for in vitro analysis. Through determination of proteolytic activity in living E. coli cells and through application of Confocal Laser-Scanning-Microscopy we demonstrate that the peptide is well suited to in vivo expression analysis. This provides an effective tool to monitor the accumulation of active recombinant TEV protease in crude extracts and intact cells.     

Improved solubility of TEV protease by directed evolution

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.     

N-terminal cysteinyl proteins can be prepared using thrombin cleavage

Expressed protein ligation--which allows native proteins to be selectively linked together by a normal peptide bond in an aqueous environment--has emerged as a powerful technique. The technique requires the formation of a C-terminal alpha-thioester and an N-terminal Cys. An N-terminal Cys can be formed by enzymatic cleavage, commonly using the Factor Xa and TEV proteases. We show that thrombin can be used for the formation of N-terminal Cys, providing another choice of reagents for expressed protein ligation. Proteins with N-terminal Cys can be obtained by the convenient modification of vectors with the putative thrombin cleavage site LVPRG to LVPRC. Two example protein domains (Csk and Abl tyrosine kinase domain) with N-terminal Cys are demonstrated using this method. The use of thrombin protease to generate N-terminal Cys overcomes some of the limitations of existing methods, making it generally useful for expressed protein ligation and other biotechnological applications.     

Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli

Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380?mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86?mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.     

Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease

Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.     

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.     

Translation-coupled translocation of yeast fumarase into mitochondria in vivo

Fumarase represents proteins that cannot be imported into mitochondria after the termination of translation (post-translationally). Utilizing mitochondrial and cytosolic versions of the tobacco etch virus (TEV) protease, we show that mitochondrially targeted fumarase harboring a TEV protease recognition sequence is efficiently cleaved by the mitochondrial but not by the cytosolic TEV protease. Nonetheless, fumarase was readily cleaved by cytosolic TEV when its import into mitochondria was slowed down by either (i) disrupting the activity of the TOM complex, (ii) lowering the growth temperature, or (iii) reducing the inner membrane electrochemical potential. Accessibility of the fumarase nascent chain to TEV protease under such conditions was prevented by low cycloheximide concentrations, which impede translation. In addition, depletion of the ribosome-associated nascent polypeptide-associated complex (NAC) reduced the fumarase rate of translocation into mitochondria and exposed it to TEV cleavage in the cytosol. These results indicate that cytosolic exposure of the fumarase nascent chain depends on both translocation and translation rates, allowing us to discuss the possibility that import of fumarase into mitochondria occurs while the ribosome is still attached to the nascent chain.     

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.

Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.     

Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast

We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site.     

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.     

Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics

Protease cleavage site recognition motifs can be identified using protease substrate discovery methodologies, but typically exhibit non‐optimal specificity and activity. To enable evolutionary optimization of substrate cleavage kinetics, a two‐color cellular library of peptide substrates (CLiPS) methodology was developed. Two‐color CLiPS was applied to identify peptide substrates for the tobacco etch virus (TEV) protease from a random pentapeptide library, which were then optimized by screening of a focused, extended substrate library. Quantitative library screening yielded seven amino acid substrates exhibiting rapid hydrolysis by TEV protease and high sequence similarity to the native seven‐amino‐acid substrate, with a strong consensus of EXLYΦQG. Comparison of hydrolysis rates for a family of closely related substrates indicates that the native seven‐residue TEV substrate co‐evolved with TEV protease to facilitate highly efficient hydrolysis. Consensus motifs revealed by screening enabled database identification of a family of related, putative viral protease substrates. More generally, our results suggest that substrate evolution using CLiPS may be useful for optimizing substrate selectivity and activity to enable the design of more effective protease activity probes, molecular imaging agents, and prodrugs. Biotechnol. Bioeng. 2010; 106: 339–346. © 2010 Wiley Periodicals, Inc.     

Genetic analysis of the short splice variant of the inhibitor of caspase-activated DNase (ICAD-S) in chicken DT40 cells

We have studied the regulation of the caspase-Activated DNase (CAD) by its inhibitor, ICAD. To study the role of ICAD short and long splice forms ICAD-S and ICAD-L, respectively, in vivo, we constructed chicken DT40 cell lines in which the entire coding regions of ICAD alone or ICAD plus CAD were deleted. ICAD and ICAD/CAD double knock-outs lacked both DNA fragmentation and nuclear fragmentation after the induction of apoptosis. We constructed a model humanized system in which human ICAD-L and CAD proteins expressed in DT40 ICAD/CAD double knock-out cells could rescue both DNA fragmentation and stage II chromatin condensation. ICAD-S could not replace ICAD-L as a chaperone for folding active CAD in these cells. However, a modified version of ICAD-S, in which the two caspase-3 cleavage sites were replaced with two tobacco etch virus (TEV) protease cleavage sites (ICAD-S(2TEV)) and which was therefore resistant to caspase cleavage, did inhibit CAD activation upon induction of apoptosis in vivo. Moreover, ICAD-L(2TEV) was functional as a chaperone for the production of active CAD in DT40 cells. In extracts prepared from these cells, we were able to activate CAD by cleavage of ICAD-L(2TEV) with TEV protease under non-apoptotic conditions. Thus, ICAD appears to be the only functional inhibitor of CAD activation in these cell-free extracts. Taken together, these observations indicate that ICAD-S may function together with ICAD-L as a buffer to prevent inappropriate CAD activation, particularly in cells where ICAD-S is the dominant form of ICAD protein.