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.     

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.     

Comparison of the substrate specificity of two potyvirus proteases

The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.     

Molecular Cloning, Expression, and Purification of Nuclear Inclusion A Protease from Tobacco Vein Mottling Virus

The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography. The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively. The K_m and k_cat values were about 0.2 mM and 0.071 s^–1, respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease. In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases. These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases.     

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.     

Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives

Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.     

Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins

The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The difficulty in obtaining this enzyme led us to look for an optimal method for its use. In this work, we produced both the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence (Streptag II-TEV(WT), and Streptag II-TEV(S219V), respectively). The two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective free forms. Both immobilized Streptag II-TEV(WT) and Streptag II-TEV(S219V) were active on the 74-kDa Streptag II substrate with a retained activity of 83.5% and 81%, respectively compared to their free corresponding forms. The slight enzyme activity decrease caused by the immobilization was balanced by the enhanced stability and the successful repetitive use of the proteolytic columns. Thus, the wild-type and the mutant immobilized proteases were used, during a period of 18 months, for nine batch reactions with retention of 38% and 51% of their initial activities, respectively. The present results demonstrate that immobilized TEV protease on streptavidin-agarose is an attractive and efficient tool for fusion protein cleavage, especially when the target protein is fused to a streptagged fusion partner. Using this strategy, the total process can be shortened by performing the cleavage and the recovery of the purified target protein in one step.     

System for cleavable Fc fusion proteins using tobacco etch virus (TEV) protease

We describe a novel Fc fusion protein system that can be cleaved by tobacco etch virus (TEV) protease. This system is desirable because it takes advantage of the high specificity of TEV protease and its activity at 4 degrees C. We produced two TEV-Fc fusion proteins that contain the first three Ig domains and all six Ig domains of the cell adhesion molecule L1. Both proteins were efficiently cleaved by TEV protease at 4 degrees C. Functional analysis of the cleavage products in neurite outgrowth assays showed they had similar activities to their parental Fc fusion proteins. Therefore, TEV-Fc fusion proteins may increase the utility and flexibility of the Fc fusion protein system.     

Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3).

Due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV protease) is a useful reagent for cleaving genetically engineered fusion proteins. However, the overproduction of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility. Here we demonstrate that the low yield can be attributed to the presence of arginine codons in the TEV protease coding sequence that are rarely used in E. coli and specifically to a tandem pair of AGA codons. The yield of protease can be improved by replacing these rare arginine codons with synonymous ones or by increasing the supply of cognate tRNA that is available to the cell. Furthermore, we show that when ribosomes become stalled at rare arginine codons in the TEV protease mRNA, the nascent polypeptides are targeted for proteolytic degradation in BL21(DE3) cells by a mechanism that does not involve tmRNA-mediated peptide tagging.     

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.     

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.     

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.     

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 Tobacco Etch Virus Protease with Increased Substrate Tolerance at the P1' position

Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein–protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1’ Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1’ Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.     

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers,Additives and Detergents Solutions for Recombinant Protein Production

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.     

采用TEV酶法进行整合膜蛋白拓扑学分析

为了研究膜蛋白的跨膜结构,进行拓扑学分析是十分重要的.有许多分析膜蛋白拓扑结构的方法,本文采用烟草蚀斑病毒（TEV）酶特异性切割测试蛋白中跨膜片段的前段或后端所插入的tev识别序列EXXYXQ(S/G),如果TEV酶能够切割,表明该序列位于目标蛋白的细胞 质外.将Tev识别序列ENLYFQG 分别插入到拟南芥整合膜蛋白的的跨膜区域,然后转化进入酿酒酵母中. 消解酶(zymolyase)酶破除酵母的细胞壁后,TEV酶消化球状体,最后通过Western免疫印迹法来分析结果.有关该方法的注意事项在结果中进行了讨论.     

Enhancing the stability and solubility of TEV protease using in silico design

The ability to rationally increase the stability and solubility of recombinant proteins has long been a goal of biotechnology and has significant implications for biomedical research. Poorly soluble enzymes, for example, result in the need for larger reaction volumes, longer incubation times, and more restricted reaction conditions, all of which increase the cost and have a negative impact on the feasibility of the process. Rational design is achieved here by means of the PoPMuSiC program, which performs in silico predictions of stability changes upon single-site mutations. We have used this program to increase the stability of the tobacco etch virus (TEV) protein. TEV is a 27-kDa nuclear inclusion protease with stringent specificity that is commonly used for the removal of solubility tags during protein purification protocols. However, while recombinant TEV can be produced in large quantities, a limitation is its relatively poor solubility (generally approximately 1 mg/mL), which means that large volumes and often long incubation times are required for efficient cleavage. Following PoPMuSiC analysis of TEV, five variants predicted to be more stable than the wild type were selected for experimental analysis of their stability, solubility, and activity. Of these, two were found to enhance the solubility of TEV without compromising its functional activity. In addition, a fully active double mutant was found to remain soluble at concentrations in excess of 40 mg/mL. This modified TEV appears thus as an interesting candidate to be used in recombinant protein technology.     

Structural basis for the substrate specificity of tobacco etch virus protease

Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2- and 1.8-A resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.     

Inhibition of tobacco etch virus protease activity by detergents

Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.     

A combined approach to improving large-scale production of tobacco etch virus protease

Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 degrees C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of approximately 400 mg/L of expression culture (approximately 15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (approximately 12 mg pure protease fusion per gram of E. coli cell paste) are also reported.