The activity of Turbo3C Protease is tested using a control target protein. 1 ug of Turbo3C Protease has at least 1 unit activity conventionally used by other suppliers (1 unit of HRV3C protease cleaves >95% of 100 ug of control target protein at 4°C for 16 hours). No non-specific activity has been observed under the same condition with Turbo3C Protease to control target protein ratio of 1:10. Prolonged incubation (several days) under the same condition does not show any non-specific cleavage.

Figure 1
A 68 kDa GST-fusion protein (C) at 1 mg/ml is incubated with Turbo3C Protease (*) at a ratio of (1) 1:50, (2) 1:100, (3) 1:200, (4) 1:400 (w/w) in a buffer of 25 mM Tris-HCl, pH8.0, 150 mM NaCl, 14 mM 2-mercaptoethanol at 4°C for 16 hours. The cleaved products are 42 kDa and 26 kDa

2 mg/ml in 50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, 50% glycerol. Store at -20°C. Shipped in gel packs.

Purified from E. coli expressing the Turbo3C protease gene.

It is recommended to use Turbo3C Protease at a protease-to-target protein ratio of 1:100 (w/w) or 1 unit of Turbo3C Protease to 100 ug of target protein in a buffer suitable for the target protein at 4°C overnight, with the target protein concentration at 1-2 mg/ml. In most cases, target proteins are completely cleaved with a protease-to-target protein ratio of 1:50 to 1:400 or 1 unit Turbo3C Protease to 50-400 ug of target protein (as shown in Figure 1). The efficiency of cleavage may vary due to the sequences around the cleavage site, the conformation and the solubility of the target protein. Due to its high specificity, more Turbo3C Protease (at 1:10 ratio) or longer cleavage time (over a weekend) at higher temperature (37°C) can be used to achieve high cleavage efficiency without non-specific cleavage of target proteins.

The Turbo3C Protease contains both GST and His tags. After cleavage of the target protein, Turbo3C Protease can be easily removed along with the tags from the cleavage reaction by affinity chromatography on a Ni-chelating resin for His-tagged target protein or GSH resin for GST-tagged target protein.

Turbo3C Procedure (PDF)