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WELQut Protease


WELQut Protease is a highly specific serine protease cleaving outside the WELQX recognition motif to remove N-terminal protein fusion tags.
  
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Thermo Scientific WELQut Protease is highly specific, recombinant serine protease of Staphylococcus aureus. It recognizes and precisely cleaves recombinant proteins containing an engineered recognition sequence† W- E- L- Q↓X (Trp, Glu, Leu, Gln, X can be any amino acid). The protease cleaves outside the recognition sequence without leaving additional amino acids bound to the target protein.

The WELQut Protease is active in a broad temperature (4 to 30°C) and pH (pH 6.5 to 9.0) range and does not require specific buffers.

In addition, this new protease has several procedural advantages - it is ideal for on-column proteolysis reactions and can be easily removed from reaction mixtures using its built-in His-tag.

Highlights

  • Cleaves outside WELQ recognition sequence, without leaving additional amino acids bound to the target protein
  • Highly specific to cognate recognition site, does not generate non-specific product bands, even after long incubation and using excess of protease
  • Easy to remove from the reaction mixture using built-in His-tag
  • Ideal for on-column proteolysis reactions

Applications

  • Removal of N-terminal fusion tags from recombinant protein preparations.

Footnote

† This cleavage sequence is present in expression vector pLATE52 included into the Thermo Scientific aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WELQut) (#K1281) available from Thermo Scientific.

  
CategoryName
Definition of Activity UnitEach unit is defined as the amount of enzyme required to cleave ≥ 99% of 100 µg of a control protein in 16 h at 20°C.
Enzyme activity is assayed in 100 µL 100 mM Tris-HCl (pH 8.0)
HazardousNo
Molecular Weight22 kDa monomer
Shelf Life
Shipping Condition
Shipping Information
SourceBacillus subtilis cells with a cloned gene of SplB protease from Staphylococcus aureus
SpecificationName
SpecificationValue
Storage BufferEnzyme is supplied in 10 mM Na2HPO4; 1.8 mM KH2PO4, pH 7.3; 140 mM NaCl; 2.7 mM KCl, 50% glycerol
Storage Condition-20 C
Stained SDS-PAGE gel showing specific cleavage products cleaved with WELQut and non-specific cleavage of target protein by competitor enterokinase.

Highly specific proteolysis of target protein performed by WELQut vs. competitor Enterokinase

Stained SDS-PAGE gel showing specific cleavage products cleaved with WELQut and non-specific cleavage of target protein by competitor enterokinase.

Figure 1 | Highly specific proteolysis of target protein performed by WELQut vs. competitor Enterokinase. Target protein (Klenow Fragment (exo-), containing WELQut or Enterokinase recognition sequence) was treated with WELQut Protease and Enterokinase according to the recommendations provided by the suppliers. Various enzyme/substrate amount ratios were tested and reaction products were analyzed in SDS-PAGE.

M - PageRuler Prestained Protein Ladder (Thermo Scientific, #SM0671).
K - Control-uncut target protein.
1-6 various WELQut/target protein amount ratios, U/µg.
1 - 1:100, 2 - 1:50, 3 - 1:40, 4 - 1:20, 5 - 1:10, 6 - 1:5.
7-11 various Enterokinase/target protein amount ratios, µg/µg.
7 - 1:100, 8 - 1:40, 9 - 1:20, 10 - 1:10, 11 - 1:5.


Stained SDS-PAGE gel showing various eluates before and after on-column protein digestion with WELQut protease

Ideal for on-column proteolysis reactions

Stained SDS-PAGE gel showing various eluates before and after on-column protein digestion with WELQut protease

Figure 2 | Ideal for on-column proteolysis reactions. Target protein (Klenow Fragment (exo-)), containing His-tag and WELQut recognition sequence, was treated with WELQut on-column, during IMAC purification procedure. (HisPur Ni-NTA Spin Columns, Thermo Scientific Pierce).

M - PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #SM0671).
1 - negative control (E.coli ER2566/pLATE52-Cat before induction).
2 - lysate load (E.coli ER2566/pLATE52-Klenow Fragment after 3 h induction with 0.1 mM IPTG.
3 – Eluate I (lane shows that after enzymatic His6 tag removal, Klenow Fragment is eluted from the IMAC sorbent).
4 – Eluate II - imidazole eluted proteins (lane shows cleavage reaction and removal of WELQut from reaction mixture efficiencies).


References

Citations