Venom Supplies PTY LTD

Cleavage Compound

Use of novel enzyme Notanarin to cleave recombinant fusion proteins in molecular biology

In molecular biology, recombinant proteins are frequently expressed fused to other proteins ("carrier" proteins), to correct purification. Cleavage sites for sequence-specific proteinases are engineered between the leader protein and the recombinant protein. Proteinases which recognize these cleavage sites are used to cleave off the leader protein. The blood coagulation proteinases factor Xa and thrombin are most frequently used. These enzymes are found in very small quantities in blood, are difficult to purify and are consequently very expensive.

Notanarin is an enzyme isolated from Notechis ater niger (Kangaroo Island South Australia). It is a 46 kDa protein consisting of two chains (30 kDa and 16 kDa) linked by a single disulfide bond. This serine proteinase is a structural homologue of blood coagulation factor Xa and is identical to it in enzymatic properties. Like factor Xa, it cleaves bonds Arg 274 - Thr 275 and Arg 323 - Ile 324 in bovine prothombin to convert it to thrombin. In addition to possessing identical sequence specificity of cleavage (Ile-Glu-Gly-Arg ) to factor Xa on prothrombin, the macromolecular physiological substrate, notanarin also specifically hydrolyzes a number of factor Xa-specific chromogenic substrates such as CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroanilide hydrochloride.

Notanarin has comparable activity to factor Xa in cleaving fusion proteins. (About 1 mg can be used to cleave 25 mg of protein). It is available in a highly purified form. It is also more stable than mammalian factor Xa. Since notanarin is as efficient as and much less expensive than factor Xa, it as an excellent alternative in molecular biology recombinant protein cleavage kits.

Unit definition: 1 unit of factor Xa cleaves 50 mg of test substrate to 95% completion in 6 hours or less. Notanarin has about 500 units per mg protein.

Assay conditions: The recombinant fusion protein is dissolved in the digestion buffer (20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl2, pH 8.0). Notanarin is added in a 1:25 (w/w) ratio. The mixture is incubated at 23ºC for 6 hours (or as long as required for complete cleavage).

Concentration and shipping: Notanarin is supplied at 0.5-1 mg/ml in a buffer containing 50% glycerol. It should be stored at -20ºC.
10 units $35
100 units $300
250 units $700
500 units $1,300

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