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Numacut TEV Protease, 6x His-Tag

The Numacut™ TEV protease is the first platform capable of producing native proteins without leaving any additional residues. Evolved by artificial intelligence and directed evolution, its recognition site from the original TEV protease (ENLYFQ↓S) has been expanded to ENLYFQ↓X (where X can be any amino acid except proline). This new recognition site enables Numacut™ to cleave target peptides or proteins fused to a C-terminal ENLYFQ, independent of the N-terminal amino acid, without leaving additional residues.

Furthermore, Numacut™ features beneficial mutations that enhance specific activity, solubility, and stability, outperforming the wild-type enzyme. The N-terminal 6x His-Tag facilitates easy and efficient removal of Numacut™ from cleavage reactions via immobilized metal-ion affinity chromatography (IMAC).
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Numacut™ allows your targets to be produced in their native form and released without any traces, such as start methionines, fusion partners, tags, cleavage scars, or cloning artifacts.

In what situations might this be important? For example, if…

… the terminus of your target is functionally important.
… the protein has stability or solubility issues with extra amino acids.
… your R&D / pre-clinical studies were performed with native proteins, and you do not want to make any changes solely for the sake of a more efficient process.
… you do not want to take any risks.

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Properties

  • Source: E. coli
  • Biological Activity: > 10,000 U/mL
  • Unit Definition: One Unit of Numacut TEV protease cleaves 80% of 3 µg control substrate containing a S amino acid residue at P1’ position in 1 h at 30°C and pH 7.5*. *Similar cleavage efficiencies observed for all amino acid residues at P1’ position except for P.
  • Molecular Weight: 28.2 kDa
  • Formulation buffer: Lyophilized from buffer (20 mM Tris/HCl buffer, 100 mM NaCl, 5 mM DTT, 1 mM EDTA, pH 7.5). No preservatives and carrier-free.
  • Purity: > 90% by SDS-PAGE analyses
  • Storage: Upon receipt, store the Numacut TEV protease at -20°C. The product is stable for at least six months. For application, reconstitute Numacut TEV protease by dissolving it with the provided reconstitution buffer. Reconstituted Numacut TEV protease should be stored at -20°C. Avoid thawing / freezing cycles.
Data Specification Sheet
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Safety Data Sheet
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Numacut TEV Protease - Case Study
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Numacut TEV Protease - Reaction Buffer - SDS
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Numacut TEV Protease - Reconstitution Buffer - SDS
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Application

10 Units of Numacut protease are recommended for the cleavage of 1-25 µg substrate (≥ 1 mg/mL) in reaction buffer (50 mM Tris/HCl, 0.5 mM EDTA and 1 mM DTT, pH 8.0). Incubate the cleavage reaction at 30°C and pH 7-8. Usually, a reaction time of 1 h is sufficient for quantitative cleavage. Increase the reaction time (≤ 24h) to improve the cleavage efficiency. Optimization of the cleavage reaction can be achieved by varying the amount of protease, reaction time and incubation temperature.

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Case Study: Numacut TEV protease

Increased substrate tolerance compared to the TEV protease wild type
The substrate tolerance of Numacut TEV protease was evaluated and compared to the wild type TEV protease. A fusion protein test substrate, featuring the classic TEV protease recognition site (ENLYFQ/S; cleavage between Q and S), was used for assay development. Subsequently, 19 additional test substrates were created, each containing the classic TEV recognition sequence with a different canonical amino acid in place of S at the P1' position. The recognition sites of the TEV protease were inserted between the target protein (located at the C-terminus) and the removable overhang protein (located at the N-terminus).

The TEV wild type and newly evolved Numacut TEV protease were added to 50 μM of test
substrate in reaction buffer (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA and 1 mM DTT), respectively, and
incubated at 30°C for 1h. Cleavage reactions were quenched by addition of 3 M GuHCl and the release of target protein from overhang protein was quantified by RP-HPLC chromatography.

The recognition site of the TEV protease (ENLYFQ/S or G) was successfully broadened to ENLYFQ/X (except for I and P) for the Numacut TEV protease. It demonstrates that the Numacut TEV protease, developed by directed evolution, has a significantly widened amino acid tolerance at the cleavage site compared to the standard TEV protease, opening the doors to unique possibilities such as:

 cleavage of overhang protein independently of the N-terminal amino acid.
 Production of native and traceless target proteins without any cleavage scars etc.
 No false by-products (due to high sequence specificity)

Please find above the downloadable PDF including further information.

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A member of the team will reach out to you with a quote within 24hrs.
For any questions beforehand, do not hesitate to send an E-Mail to
products@numaferm.com or call us on +49 211 975 32 900.
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