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Purification & Detection

Epitope Tag Removal

Epitope tagging has become a powerful tool for the detection and purification of expressed proteins. Presence of an epitope tag in the recombinant protein can raise concerns about the effect of additional amino acid residues on the structure and physical properties of the protein. To avoid these problems, epitope tags may be removed by enzymatic methods resulting in purified proteins containing little or no residual epitope tag sequence. Sigma-Aldrich offers a range of enzymes and kits to remove the epitope tag from your target protein for easier downstream analysis.


Enterokinase
Product No.

Description

Recognition Peptide

E5144

Highly specific serine protease used for the removal of the FLAG® peptide from fusion proteins. It is supplied as a Na Cl form, lyophilized from deionized water.

LYS-X of FLAG
N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-X-C
Note: Peptides are resistant to cleavage if proline occupies position X.

 
Enterokinase Removal Kit
Product No.

Description

Recognition Peptide

PRKE

Designed as research tool for the removal of bovine enterokinase from mixtures containing a fusion protein cleaved by the enzyme. Using the kit, removal of essentially all enterokinase is accomplished by binding with immobilized rabbit antibodies to calf intestine enterokinase followed by spin filtration.
Anti-Enterokinase-Agarose Conjugate, 1.5 mL
20x Wash Buffer, 4 mL
Spin Filters, 10 each

LYS-X of FLAG
N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-X-C
Note: Peptides are resistant to cleavage if proline occupies position X.

 
TEV Protease
Product No.

Description

Recognition Peptide

T4455 TEV protease, originally isolated from tobacco etch virus, is a highly site-specific cysteine protease that recognizes the sequence Glu-P4-P3-Tyr-P2-Gln-Gly/Ser. TEV protease selectively cleaves between the Gln and the Gly/Ser sites. The most common such sequence is ENLYFQS. Glu-P4-P3-Tyr-P2-Gln-Gly/Ser
(e.g. Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser)
 
Thrombin CleanCleave™ Kit
Product No.

Description

Recognition Peptide

RECOMT

Bovine thrombin immobilized on 4% beaded agarose designed for cleavage of recombinant fusion proteins. This format circumvents the need for removal of thrombin by chromatographic techniques. The ligand density allows for fast and efficient cleavage; the resin can be reused multiple times with only minimal loss in cleavage efficiency.

P4-P3-Pro-Arg/Lys-X-P1'-P2'
Note: P4 and P3 are hydrophobic residues, P1' and P2' are non-acidic residues, and Arg/Lys-X-P1' is the scissile bond.
P2-Arg/Lys-X-P1'
Note: Where P2 or P1' is glycine and Arg/Lys-X-P1' is the scissle bond.

 
Thrombins
Product No.

Description

Recognition Peptide

T1063

T7513

Thrombin is an endolytic serine protease that selectively cleaves the Arg--Gly bonds. The soluble forms of thrombin are also used for cleavage of recombinant fusion proteins. Human and bovine thrombins have the same peptide recognition sequence.

P4-P3-Pro-Arg/Lys-X-P1'-P2'
Note: P4 and P3 are hydrophobic residues, P1' and P2' are non-acidic residues, and Arg/Lys-X-P1' is the scissile bond.
P2-Arg/Lys-X-P1'
Note: Where P2 or P1' is glycine and Arg/Lys-X-P1' is the scissle bond.

F9302

Activated Factor X (Xa) is used for cleavage of recombinant fusion protein tags in applications similar to thrombin. Factor Xa catalyzesthe hydrolysis of Arg-Thr and Arg-Ile bonds.

The fairly strict recognition sequence is Ile-Glu (or Asp)-Gly-Arg--X. It maysometimes cleave at other basic residues, depending on the conformation of the target protein. Factor Xa will not cleave if a proline residue follows the arginine of the recognition sequence.

 
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