TEV protease-mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms

Drosophila provides a powerful experimental system to analyze gene functions in a multi-cellular organism. Here we describe an in vivo method that interferes with the integrity of selected proteins through site-specific cleavage in Drosophila. The technique is based on the highly specific seven-amino-acid recognition site of the tobacco etch virus (TEV) protease. We established transgenic fly lines that direct TEV protease expression in various tissues without affecting fly viability. The insertion of the TEV protease recognition site in defined positions of target proteins mediates their sequence-specific cleavage after controlled TEV protease expression in the fly. Thereby, this technique is a powerful tool that allows the in vivo manipulation of selected proteins in a time- and tissue-specific manner.     

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.     

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 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.     

Biochemical and mutational analysis of a plant virus polyprotein cleavage site.

The RNA genome of tobacco etch virus (TEV) is organized as a single translational unit coding for a 346,000 (346 kd) mol. wt (Mr) polyprotein. The 346 kd Mr polyprotein is cleaved by a 49 kd Mr virus-encoded proteinase at five different sites between the dipeptides Gln-Ser or Gln-Gly. These cleavage sites or gene product boundaries are defined by the heptapeptide sequence...Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly.... We have used the 54 kd Mr nuclear inclusion protein/30 kd Mr capsid protein junction as a model to examine the role of these conserved amino acids in defining a cleavage site. The 54 kd/30 kd Mr protein cleavage site sequence of 10 TEV isolates from geographically distinct locations has been deduced. The conserved amino acids are present in all isolates. To determine if these four amino acids are an absolute requirement for polyprotein substrate activity, a site-directed mutational analysis has been performed. A recombinant cDNA molecule encoding the TEV 54 kd/30 kd Mr gene product cleavage site was mutated and polyprotein substrates were synthesized and processed in a cell-free system. Single amino acid substitutions made at the different positions reveal a strong preference for the naturally conserved amino acids.     

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.     

A "two-hit" hypothesis for inclusion formation by carboxyl-terminal fragments of TDP-43 protein linked to RNA depletion and impaired microtubule-dependent transport

Carboxyl-terminal fragments (CTFs) of TDP-43 aggregate to form the diagnostic signature inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the biological significance of these CTFs and how they are generated remain enigmatic. To address these issues, we engineered mammalian cells with an inducible tobacco etch virus (TEV) protease that cleaves TDP-43 containing a TEV cleavage site. Regions of TDP-43 flanking the second RNA recognition motif (RRM2) are efficiently cleaved by TEV, whereas sites within this domain are more resistant to cleavage. CTFs containing RRM2 generated from de novo cleavage of nuclear TDP-43 are transported to the cytoplasm and efficiently cleared, indicating that cleavage alone is not sufficient to initiate CTF aggregation. However, CTFs rapidly aggregated into stable cytoplasmic inclusions following de novo cleavage when dynein-mediated microtubule transport was disrupted, RNA was depleted, or natively misfolded CTFs were introduced into these cells. Our data support a "two-hit" mechanism of CTF aggregation dependent on TDP-43 cleavage.     

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.     

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.     

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.     

Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.     

Overview and analysis of the polyprotein cleavage sites in the family Potyviridae

The genomes of plant viruses in the family Potyviridae encode large polyproteins that are cut by virus-encoded proteases into ten mature proteins. Three different types of protease have been identified, each of which cuts at sites with a distinctive sequence pattern. The experimental evidence for this specificity is reviewed and the cleavage site patterns are compiled for all sequenced species within the family. Seven of the nine cleavage sites in each species are cut by the viral NIa-Pro and patterns around these sites are related where possible to the active site–substrate interactions recently deduced following the resolution of the crystal structure of Tobacco etch virus (TEV) NIa-Pro (Phan et al ., 2002. J. Biol. Chem . 277, 50564–50572). In particular, a revised series of cleavage sites for Sweet potato mild mottle virus (genus Ipomovirus ) is proposed with a conserved His at the P1 position. This is supported by homology modelling studies using the TEV structure as a template. The data also provide a standard to correct the annotation of some other published sequences and to help predict these sites in further virus sequences as they become available. Comprehensive data for all sequences of each virus in the family, together with some summaries, have been made available at http://www.rothamsted.bbsrc.ac.uk/ppi/links/pplinks/potycleavage/index.html .     

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages.

Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate.     

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.     

Structural determinants of tobacco vein mottling virus protease substrate specificity

Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac‐RETVRFQSD) at 1.7‐Å resolution. As observed in several crystal structures of TEV protease, the C‐terminus (～20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ～10‐fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1′ position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k_cat and K_m for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.     

Exosite interactions determine the affinity of factor X for the extrinsic Xase complex

The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VIIa within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X. This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical noncompetitive inhibition of factor X activation by active site-directed ligands. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protein substrate affinity, whereas the second binding step influences the maximum catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with exosite binding using factor X derivatives that are expected to have limited or abrogated interactions with the active site of VIIa within Xase. Thus, substrate interactions at exosites, sites removed from the active site of VIIa within the enzyme complex, determine affinity and binding specificity in the productive recognition of factor X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homologous enzymes of coagulation.     

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.     

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.     

Internal core protein cleavage leaves the hepatitis B virus capsid intact and enhances its capacity for surface display of heterologous whole chain proteins

Virus capsids find increasing use as nanoparticulate platforms for the surface display of heterologous ligands, including as multivalent vaccine carriers. Presentation on the icosahedral hepatitis B virus capsid (HBcAg) is known to strongly enhance immunogenicity of foreign sequences, most efficiently if they are inserted into the dominant c/e1 B cell epitope, a surface-exposed loop in the center of the constituent core protein primary sequence. Even some complete proteins were successfully inserted but others, e.g. the outer surface protein A (OspA) of the Lyme disease agent Borrelia burgdorferi, impaired formation of capsid-like particles (CLPs). This difference can be rationalized by the requirement for the termini of the insert to fit into the predetermined geometry of the two acceptor sites in the carrier. We reasoned that cleavage of one of the two bonds connecting insert and carrier should relieve these constraints, provided the cleaved protein fragments remain competent to support the particle structure. Indeed, HBcAg CLPs containing a recognition site for tobacco etch virus (TEV) protease in the c/e1 loop remained intact after cleavage, as did CLPs carrying a 65-residue peptide insertion. Most importantly, in situ cleavage of a core-OspA fusion protein by coexpressed TEV protease strongly enhanced CLP formation compared with the uncleaved protein. These data attest to the high structural stability of the HBcAg CLP and they significantly widen its applicability as a carrier for heterologous proteins. This approach should be adaptable to any protein-based particle with surface-exposed yet sequence-internal loops.