Download Protease 3C, GST-tagged (data sheet)

GST - Protease 3C (PreScission® Protease)
Source
The used Protease 3C variant is encoded by the human rhinovirus species of serotype 14.
Description
The used variant is genetically fused with an N-terminal Glutathion-S-Transferase affinity tag (GST)
resulting in a fusion protein of approximately 46 kDa. The Protease 3C belongs to the class of
cysteine proteases and recognizes Leu-Glu-Val-Leu-Phe-Gln|Gly-Pro (L-E-V-L-F-Q|G-P) as cleavage
motif. The cleavage occurs between the amino acids Gln (Q) and Gly (G)[1] like in the commercial
available pGEX-6P(1-3) vector series from GE Healthcare that contains a recognition site for cleaving
the target protein from the fusion construct[2].
Purification
The GST - Protease 3C was purified until homogeneity with GSHaffinity chromatography and subsequent size-exclusion
chromatography as polishing step. The resulting protein showed a
purity of more than 95% (Figure 1).
120
85
50
GST - Protease 3C
35
25
20
Figure 1: Electrophoretic analysis of
1 µg purified GST - Protease 3C.
Activity assay
M
1
2
3
4
5
6
7
8
9
All cleavage reactions with GST - Protease 3C are flexible
regarding the used incubation temperature. GST Protease 3C showed proteolytic activity when used at room
temperature or at 4 °C. The following example shows the
cleavage efficiency of 0,25 µg GST - Protease 3C on 100 µg
substrate at 4 °C (Figure 2).
Figure 2: Cleavage of 100 µg single-step
purified GST-YscP (77 kDa) with 0,25 µg
GST -Protease 3C at 4 °C. (1) t=0’, (2) t=10’,
(3) t=30’, (4) t=60’, (5) t=1,5h, (6) t=2h, (7)
t=3h, (8) t=4h, (9) t=6h
calculated specific activity (U/mg*1):
available as:
42 U/mg
1 ml aliquots each 2 mg/ml
(sufficient to cleave 800 mg of 77 kDa target protein)
*1
specific activity, U/mg: amount substrate (µmol) digested per minute by 1 mg enzyme or protease
References
[1]
M. G. Cordingley, P. L. Callahan, V. V. Sardana, V. M. Garsky and R. J. Colonno, “Substrate
Requirements of Human Rhinovirus 3C Protease for Peptide Cleavage in Vitro”, The Journal of
Biological Chemistry, vol. 265, no. 16, pp. 9062-9065, June 1990
[2]
GE Healthcare, http://www.gelifesciences.com