TEV Protease

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David S Waugh - One of the best experts on this subject based on the ideXlab platform.

  • Removal of affinity tags with TEV Protease
    Methods in Molecular Biology, 2017
    Co-Authors: Sreejith Raran-kurussi, Simon Cherry, Di Zhang, David S Waugh
    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.

  • expression and purification of soluble his 6 tagged TEV Protease
    Methods of Molecular Biology, 2009
    Co-Authors: Joseph E. Tropea, Scott Cherry, David S Waugh
    Abstract:

    This chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (TEV) Protease in Escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. The Protease is initially produced as a fusion to the C-terminus of E. coli maltose binding protein (MBP), which causes it to accumulate in a soluble and active form rather than in inclusion bodies. The fusion protein subsequently cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV Protease catalytic domain with an N-terminal polyhistidine tag. The His-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 gives rise to an enzyme with greater catalytic efficiency than the wild-type Protease.

  • hexahistidine tagged maltose binding protein as a fusion partner for the production of soluble recombinant proteins in escherichia coli
    Methods of Molecular Biology, 2009
    Co-Authors: Brian P Austin, Sreedevi Nallamsetty, David S Waugh
    Abstract:

    Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinatorially-tagged His(6)MBP fusion proteins by recombinational cloning and how to evaluate their yield and solubility. We also describe a procedure to determine how efficiently a His(6)MBP fusion protein is cleaved by tobacco etch virus (TEV) Protease in E. coli and a method to assess the solubility of the target protein after it has been separated from His(6)MBP.

  • efficient site specific processing of fusion proteins by tobacco vein mottling virus Protease in vivo and in vitro
    Protein Expression and Purification, 2004
    Co-Authors: Sreedevi Nallamsetty, Terry D. Copeland, Rachel B Kapust, Joseph E. Tropea, Scott Cherry, József Tözsér, David S Waugh
    Abstract:

    AYnity 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 speciWcity. 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 speciWcity. The tobacco vein mottling virus (TVMV) Protease is a close relative of TEV Protease with a distinct sequence speciWcity (ETVRFQG/S). We show that, like TEV Protease, TVMV Protease can be used to cleave fusion proteins with high speciWcity 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 diVer only two-fold in catalytic eYciency, both at room temperature and 4 °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. Published by Elsevier Inc.

  • processive degradation of nascent polypeptides triggered by tandem aga codons limits the accumulation of recombinant tobacco etch virus Protease in escherichia coli bl21 de3
    Protein Expression and Purification, 2002
    Co-Authors: Rachel B Kapust, Karen M. Routzahn, David S Waugh
    Abstract:

    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.

Jun Fan - One of the best experts on this subject based on the ideXlab platform.

  • construction investigation and application of the TEV Protease variants with the improved oxidative stability
    Journal of Microbiology and Biotechnology, 2021
    Co-Authors: Enkhtuya Bayar, Yuanyuan Ren, Yinghua Chen, Shuncheng Zhang, Jun Fan
    Abstract:

    Tobacco etch virus Protease (TEVp) is a useful tool for removing fusion tag, but wild type TEVp is less stable under the oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into the TEVp variant containing T17S, L56V, N68D, I77V and S135G for improving protein solubility, and S219V for inhibiting self-proteolysis. The solubility and cleavage activity of the constructed variants in E. coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to the improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with the enhanced yields was released from the regenerated amorphous cellulose via the affinity absorption of the cellulose-binding module as the affinity tag.

  • combination of the mutations for improving activity of TEV Protease in inclusion bodies
    Bioprocess and Biosystems Engineering, 2021
    Co-Authors: Yinghua Chen, Yuanyuan Ren, Wenjun Xiao, Jun Fan
    Abstract:

    Tobacco etch virus Protease (TEVp) is an enzymatic reagent to remove fusion tag, but additional purification steps are required for removing the TEVp after cleavage reaction is finished. Use of carrier-free and dependent TEVp immobilizates can eliminate Protease contamination. In this work, we identified that, among the four constructed missense variants, the insoluble variant with the highest activity was correspondent with the soluble one tested formerly. The activities of the insoluble 15 codon variants were assayed and the variant with highest activity was selected. The K45F and/or E106G mutations have been reported on slightly improving protein stability of the wild-type TEVp, but only E106G mutation enhanced soluble production and activity of the selected TEVp variant, and it increased soluble amounts of two codon variants with the impaired folding. The decreased activity and use efficiency of the optimized TEVp variant in inclusion bodies was balanced by the determined high level production, lower leaking amounts of the protein, the enhanced resistance to the limited proteolysis mediated by Protease K and trypsin, and the increased inhibition of auto-cleavage, as comparison to those of the immobilized soluble one. Thus, the TEVp construct is a potential alternate for simplifying protein purification protocols after tag-removal.

  • cleavage of fusion proteins on the affinity resins using the TEV Protease variant
    Protein Expression and Purification, 2017
    Co-Authors: Kejun Zhu, Beijiu Cheng, Xuan Zhou, Yanping Yan, Yifan Xie, Jun Fan
    Abstract:

    It is documented that the tobacco etch virus Protease (TEVp) variant TEVp3M is less efficient in cleaving the fusion protein bound to Ni-NTA resin at relatively low temperature. Here, we determined that, using the GFP fusion substrate bound to Ni-NTA or Strep-tactin agarose, activity of the TEVp5M variant was higher than that of the other TEVp construct, and about 15% higher than that of the TEVp3M. The GST fusion proteins immobilized on Strep-tactin agarose or Glutathione Sepharose were efficiently cleaved by purified TEVp5M at specified conditions using GFP reporter for visual track and detection. After on-column cleavage of three fusion constructs using the cognate TEVp5M constructs, two target proteins with relatively high purity were separated from Ni-NTA or Amylose agarose. With elution of the buffer containing 1 M NaCl, maize sulfiredoxin was released from Ni-NTA resin via on-column cleavage. Our results confirmed that TEVp5M efficiently cleaved the fusion proteins bound to the four affinity matrices. By combination with appropriate affinity handles, the cognate TEVp5M mediating tag removal enabled purification and cleavage of the fusion proteins, removal of the Protease, and separation of the target proteins from the affinity resin to be accomplished in one step.

  • Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli
    Applied biochemistry and biotechnology, 2016
    Co-Authors: Jiaqi Sun, Weiyu Wang, Li Jiang, Beijiu Cheng, Jun Fan
    Abstract:

    In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) Protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the Protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

  • Synonymous Rare Arginine Codons and tRNA Abundance Affect Protein Production and Quality of TEV Protease Variant
    PloS one, 2014
    Co-Authors: Jie Fang, Beijiu Cheng, Lingling Zou, Xuan Zhou, Jun Fan
    Abstract:

    It has been identified that a TEV Protease (TEVp) variant, TEVp5M, displays improved solubility. Here, we constructed fifteen TEVp5M variants with one or more of six rare arginine codons in the coding sequence replaced with abundant E. coli arginine codons. These codon variants expressed in either E. coli BL21 (DE3) or Rossetta (DE3) showed different solubility and activity. Supply of rare tRNAs increased the tendency of certain codon variants to form insoluble aggregates at early induction stage, as determined by the fused S-tag. About 32% increase in soluble protein production of M5 variant with four synonymously mutated arginine codons was identified in Rossetta (DE3) cells using GFP fusion reporter, comparable to that of TEVp5M. After purification, two other codon variants from both E. coli strains exhibited less activity than TEVp5M on cleaving the native or modified recognition sequence incorporated between GST and E. coli diaminopropionate ammonialyase by enzyme-coupled assay, whereas purified M5 variant showed activity similar to the TEVp5M. Supply of rare tRNAs caused the decrease of activity of TEVp5M and M5 by about 21%. Our results revealed that engineering of highly soluble TEVp variants can be achieved by the combined mutations of amino acid residues and optimization of specific rare codons, whereas simple augment of rare tRNAs abundance resulted in partial loss of activity.

Jin Liu - One of the best experts on this subject based on the ideXlab platform.

  • A Genetically Encoded, Phage-Displayed Cyclic-Peptide Library.
    Angewandte Chemie (International ed. in English), 2019
    Co-Authors: Xiaoshan Shayna Wang, Peng-hsun Chase Chen, J Trae Hampton, Jeffery M Tharp, Catrina A Reed, Sukant K Das, Duen-shian Wang, Hamed S. Hayatshahi, Yang Shen, Jin Liu
    Abstract:

    Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic-peptide ligands for therapeutic targets, phage-displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage-display technique in which its displayed peptides are cyclized through a proximity-driven Michael addition reaction between a cysteine and an amber-codon-encoded Nϵ -acryloyl-lysine (AcrK). Using a randomized 6-mer library in which peptides were cyclized at two ends through a cysteine-AcrK linker, we demonstrated the successful selection of potent ligands for TEV Protease and HDAC8. All selected cyclic peptide ligands showed 4- to 6-fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.

Martin Grumet - One of the best experts on this subject based on the ideXlab platform.

  • system for cleavable fc fusion proteins using tobacco etch virus TEV Protease
    BioTechniques, 2001
    Co-Authors: Jeffrey Haspel, Cy Blanco, Jeffrey Jacob, Martin Grumet
    Abstract:

    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.

Xiaoshan Shayna Wang - One of the best experts on this subject based on the ideXlab platform.

  • A Genetically Encoded, Phage-Displayed Cyclic-Peptide Library.
    Angewandte Chemie (International ed. in English), 2019
    Co-Authors: Xiaoshan Shayna Wang, Peng-hsun Chase Chen, J Trae Hampton, Jeffery M Tharp, Catrina A Reed, Sukant K Das, Duen-shian Wang, Hamed S. Hayatshahi, Yang Shen, Jin Liu
    Abstract:

    Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic-peptide ligands for therapeutic targets, phage-displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage-display technique in which its displayed peptides are cyclized through a proximity-driven Michael addition reaction between a cysteine and an amber-codon-encoded Nϵ -acryloyl-lysine (AcrK). Using a randomized 6-mer library in which peptides were cyclized at two ends through a cysteine-AcrK linker, we demonstrated the successful selection of potent ligands for TEV Protease and HDAC8. All selected cyclic peptide ligands showed 4- to 6-fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.