A new tagged-TEV protease: Construction, optimisation of production, purification and test activity

Miladi, Baligh; Bouallagui, Hassib; Dridi, Cyrine; Marjou, Ahmed El; Boeuf, Guilhem; Martino, Patrick Di; Dufour, Florence; Elm’selmi, Abdellatif

doi:10.1016/j.pep.2010.08.012

Cited by 27 publications

(9 citation statements)

References 32 publications

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“…They were then purified again over an anion–exchange column to yield nearly pure protein. The N-terminal 6× His purification tag was cleaved by incubation with TEV ( t obacco e tch v irus) protease [48], yielding native RI proteins with a single N-terminal glycine residue. Molecular masses of RI proteins were confirmed by MALDI–TOF mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles

Lomax

Bianchetti

Chang

et al. 2014

Journal of Molecular Biology

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Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species, but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92 Å, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen-sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.

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Section: Methodsmentioning

confidence: 99%

Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles

Lomax

Bianchetti

Chang

et al. 2014

Journal of Molecular Biology

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“…Further improvements in the production of soluble His-tagged TEV protease were subsequently realized, yielding up to 400 mg/L or 15 mg/g of cell paste [58,59]. TEV protease has also been fused to a number of other affinity tags, including glutathione S-transferase [59], maltose binding protein (MBP) [56], and the Streptag II [60]. TEV protease expression vectors can be obtained from a variety of sources, including the American Type Culture Collection (ATCC), the Addgene plasmid repository (http:// www.addgene.org), and the Protein Structure Initiative Biological Materials Repository (http://psimr.asu.edu/).…”

Section: Tev Proteasementioning

confidence: 99%

An overview of enzymatic reagents for the removal of affinity tags

Waugh

2011

Protein Expression and Purification

303

224

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Although they are often exploited to facilitate the expression and purification of recombinant proteins, every affinity tag, whether large or small, has the potential to interfere with the structure and function of its fusion partner. For this reason, reliable methods for removing affinity tags are needed. Only enzymes have the requisite specificity to be generally useful reagents for this purpose. In this review, the advantages and disadvantages of some commonly used endo- and exoproteases are discussed in light of the latest information.

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“…Wang et al (1997) showed that purified 3C protease had a k cat /K m value of 840 M À1 s À1 for the substrate EALFQ-pNA. Alternatively, kinetics studies by Miladi et al (2011) showed a much lower k cat /K m (260 M À1 s…”

Section: Resultsmentioning

confidence: 99%

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

Bryan

Bhandari

Napuli

et al. 2011

Acta Crystallogr F Struct Biol Cryst Commun

115

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The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two Ä KTAexplorer 100s and four Ä KTAprimes to perform immobilized metalaffinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography.

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A new tagged-TEV protease: Construction, optimisation of production, purification and test activity

Cited by 27 publications

References 32 publications

Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles

Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles

An overview of enzymatic reagents for the removal of affinity tags

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

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