Search results for: Caspase 8 Substrate IETD pNA
#17658284 2007/06/26 To Up
Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures.
IChee Yong Yun, Sen Liu, Sing Fee Lim, Tianhua Wang, Beatrice Y F Chung, Joong Jiat Teo, Kok Hwee Chuan, Allyson S C Soon, Keng Siong Goh, Zhiwei Song
2673 related Products with: Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures.
1.00 flask96 tests10 ug1 mg1x10e7 cells1.00 flask-96 wells1x10e7 cells1 mgRelated Pathways
-
No related Items
#11888206 // To Up
Expression, preparation, and high-throughput screening of caspase-8: discovery of redox-based and steroid diacid inhibition.
Because of the intimate role of caspase-8 in apoptosis signaling pathways from FAS, TNFR1, and other death receptors, the enzyme is a potentially important therapeutic target. We have generated an Escherichia coli expression construct for caspase-8 in which a His-tag sequence is inserted ahead of codon 217 of caspase-8. The strain produced a significant amount of soluble His-tagged 31-kDa inactive single-chain enzyme precursor. This 31-kDa protein could be purified to 98% purity. Hydroxyapatite resolved the enzyme into two species, one with the appropriate 31,090 relative mass and the other with 178 units additional mass. The latter proved to result from E. coli-based modification of the His-tag with one equivalent of glucono-1,5-lactone. The purified proteins could be activated by autoproteolysis to the appropriate 19- plus 11-kDa enzyme by the addition of dithiothreitol in appropriate buffer conditions. This yielded an enzyme with specific activity of 4-5 units/mg against 200 microM Ac-IETD-pNA at 25 degrees C. The fully active protein was used in a high-throughput screen for inhibitors of caspase-8. A preliminary robustness screen demonstrated that caspase-8 is susceptible to reactive oxygen-based inactivation in the presence of dithiothreitol (DTT) but not in the presence of cysteine. Investigation into the mechanism of this inactivation showed that quinone-like compounds were reduced by DTT establishing a reactive oxygen generating redox cycle the products of which (likely H(2)O(2)) inactivated the enzyme. A new class of caspase-8 inhibitors, steroid-derived diacids, with affinity in the low micromolar range were discovered in the refined screen. Structure--activity investigation of the inhibitors showed that both the steroid template and the acid moieties were required for activity.Gary K Smith, David G Barrett, Kevin Blackburn, Michael Cory, Walter S Dallas, Roderick Davis, Daniel Hassler, Randy McConnell, Mary Moyer, Kurt Weaver
2636 related Products with: Expression, preparation, and high-throughput screening of caspase-8: discovery of redox-based and steroid diacid inhibition.
100 assays25 mg4 Sample Kit10 mg100ul500 MG25 mg2 Sample Kit100ug96T100ulRelated Pathways
#10733893 // To Up
Caspase 8: an efficient method for large-scale autoactivation of recombinant procaspase 8 by matrix adsorption and characterization of the active enzyme.
A gene coding for a truncated form of human procaspase 8 has been cloned and expressed in Escherichia coli. This construct contains M(206) through D(479) of human procaspase 8, preceded by an N-terminal polyhistidine tag. The recombinant protein, containing 286 amino acids, was expressed in high yield in the form of inclusion bodies (IB). The IB were solubilized in guanidinium chloride and dialyzed against 50% acetic acid. The solution was mixed with 9 volumes of H(2)O and then rapidly diluted from the acidic medium to one containing 1.0 M Tris, pH 8.0, and 5 mM DTT. SDS-PAGE analysis of the soluble, dilute protein solution (20-30 microgram of protein/ml) showed a single 33-kDa band corresponding to the nonprocessed, inactive procaspase 8. Concentration of the dilute protein to levels as high as 2 mg/ml resulted in only modest (1-10%) autocatalytic conversion to the 19- and 11-kDa polypeptide subunits which are characteristic of the activated enzyme. Further concentration of these protein solutions to a near-dry state on the ultrafiltration membrane, followed by washing of the membrane with buffer, led to extracts containing high yields of enzyme showing a specific activity of 8.43 micromol/min/mg against the chromogenic substrate Ac-IETD-pNA. SDS-PAGE, protein sequencing, and mass spectrometric analysis of these extracts showed complete conversion of the 33-kDa procaspase 8 to the 19- and 11-kDa subunits of activated caspase 8. This method allows for preparation of 100-mg quantities of highly pure and active recombinant human caspase 8. Enzyme activity was shown to be associated with a heterotetrameric complex that is converted to an inactive dimer upon storage.K A Koeplinger, A M Mildner, J W Leone, J S Wheeler, R L Heinrikson, A G Tomasselli
2963 related Products with: Caspase 8: an efficient method for large-scale autoactivation of recombinant procaspase 8 by matrix adsorption and characterization of the active enzyme.
100 100 5 G100 100 100ul100 units100 units100 ugLabels 2 mg of Ab100 µg100ugRelated Pathways
Contact Us:
Belgium
Voortstraat 49, 1910 Kampenhout BELGIUM
Tel 0032 16 58 90 45 Fax 0032 16 50 90 45
[email protected]
France
9, rue Lagrange, 75005 Paris
Tel 01 43 25 01 50 Fax 01 43 25 01 60
[email protected]
Germany
GENTAUR GmbH
Marienbongard 20
52062 Aachen Deutschland
Tel 0241 40 08 90 86 Fax 0241 55 91 05 36
[email protected]
United Kingdom
GENTAUR Ltd.
Howard Frank Turnberry House
1404-1410 High Road
Whetstone London N20 9BH
Tel 020 3393 8531 Fax 020 8445 9411
[email protected]
Also in
Luxembourg +35220880274
Schweiz Züri +41435006251
Danmark +4569918806
Österreich +43720880899
Česká republika Praha +420246019719
Ireland Dublin +35316526556
Norge Oslo +4721031366
Finland Helsset +358942419041
Sverige Stockholm +46852503438
Ελλάς Αθήνα +302111768494
Magyarország Budapest +3619980547
Poland
GENTAUR Poland Sp. z o.o.
ul. Grunwaldzka 88/A m.2
81-771 Sopot, Poland
Tel 058 710 33 44
Fax 058 710 33 48
[email protected]
skype gentaurpoland
Nederland
GENTAUR Nederland BV
Kuiper 1
5521 DG Eersel Nederland
Tel 0208-080893 Fax 0497-517897
[email protected]
Italy
GENTAUR SRL
IVA IT03841300167
Piazza Giacomo Matteotti, 6, 24122 Bergamo
Tel 02 36 00 65 93 Fax 02 36 00 65 94
[email protected]
Spain
GENTAUR Spain
Tel 0911876558
[email protected]
Bulgaria
GENTAUR Bulgaria
53 Iskar Str. 1191 Kokalyane, Sofia
Sofia 1000
Tel 0035924682280
Fax 0035929830072
[email protected]