Numacut TEV Protease, 6x His-Tag

The Numacut TEV protease is the first protease platform to produce native proteins. Evolved by applying artificial intelligence and directed evolution, the recognition site from TEV protease (ENLYFQ/S) was broadened to ENLYFQ/X (X: any amino acid, except P). Because of this new recognition site, your target is fused C-terminal to ENLYFQ and is cleaved-off by Numacut TEV protease independently of the N-terminal amino acid.

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This way your targets are produced natively and are released traceless i.e. from Start-Met, fusion partners, tags, cleavage scars or cloning artefacts.

In which cases this might be important? For example, if…

… the terminus of your target is functionally important.
… the the protein has stability or solubility issues with the additional amino acid.
… your R&D/preclinical studies were performed with native proteins, and you do not want to change anything “just” for a more efficient process.
… you do not want to take any risks.

All beneficial mutations from Numaferm’s TEV protease are conserved, making the Numacut TEV protease the ideal sequence-specific protease platform for native protein production.
Our Numacut TEV protease harbors an N-terminal 6x His-Tag for easy removal by immobilized metal-ion affinity chromatography (IMAC).

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 (pH 8.0), 0.5 mM EDTA and 1 mM DTT). Incubate the cleavage reaction between 30-37°C and at pH 7-8. Usually, a reaction time of 1h is sufficient for quantitative cleavage. Increase the reaction time (≤ 24h) to improve the cleavage efficiency. Avoid > 0.5 M urea or 0.5 M GuHCl, pH values below 6 and above 9, and high salt concentrations ( > 150 mM). Optimization of the cleavage reaction can be achieved by varying the amount of protease, reaction time and incubation temperature.

Numacut TEV protease - Case Study

Increased substrate tolerance compared to the TEV protease wildtype

The substrate tolerance of Numacut TEV protease was assessed and compared to the TEV protease
wildtype. A test substrate consisting of a fusion protein, bearing the classic recognition site of TEV protease (ENLYFQ/S; cleavage between Q & S)) between both the fused proteins was used for the assay
development. Afterwards, 19 additional test substrates were generated, containing the classic TEV recognition sequence and instead of S (P1’ position), all additional canonical amino acids.
The recognition sites of the TEV protease were inserted between the target protein (located at the Cterminus) and the overhang protein was removed (located at the N-terminus).

Numaferm’s standard TEV protease and 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 3M guanidium hydrochloride
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.

Numacut TEV Protease, 6x His-Tag

Properties

Application

Numacut TEV protease - Case Study

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