IGEM:IMPERIAL/2009/Assays Protocols/Trehalose/OtsA
OtsA : Trehalose –6-Phosphate synthase
TPS Assay : A quantitative assay of the activity of Trehalose –6-Phosphate synthase (units of activity given in μmol of product per minute).
Shopping List
UDPG
G-6-P
Tris-HCl Buffer
MnCl2
heparin salt
HCl
NaOH
anthrone
Shodex Sugar
Alternative Assay
Trehalose –6-Phosphate synthase assay : non-radioative method Adapted from Vandercammen et al (Eur. J. Biochem. 182, 613-620., 1989)
Reaction:
Tre6Psynthase:
UDPglucose + Glucose-6-phosphate => Trehalose-6P + UDP
Enzymatic coupling:
Pyruvate kinase: UDP + PEP => Pyruvate + UTP
Lactate Dehydrogenase: Pyruvate + NADH => Lactate + NAD
Preparation of crude extract:
- Break open 15-20 mg dry mass (109 cells) in 0.5 ml extraction buffer (Hepes 20 mM, pH 7.1, DTT 1mM, KCl 100 mM; cocktail inhibitor from Boerhinger)/ 1 g glass beads by vortexing 6 times 30 sec with 30 sec interval in ice.
- Centrifuge tubes at 3000g for 5 min at 4°C
- transfer supernatant into eppendorf tube
- Centrifuge 15min in eppendorf centrifuge at full speed at 4°C
Enzymatic assay:
In 1 ml final volume
Mixture TPS 0.5 ml
(Hepes 50 mM, pH 7.1, KCl 100 mM; EDTA 2mM, MgSO4 5 mM)
UDPglucose 20 mM 0.1 ml
Glc-6-P/ F-6-P 100 mM/30 mM 0.1 ml
Water 0.2 ml
Sample 0.1 ml
Incubate at 42°C
After 10, 20, 30 min
Take 0.25 ml in 0.75 ml of ice-cold UDP mixture kept in ice-cold water bath
Measure OD340 (E0)
Add Lactate dehydrogenase 100 U/ml 1 µl
Read OD340 (E1)
Add Pyruvate kinase 100U/ml 1 µl
Read OD340 (E2)
Calcul of activity:
Tre6PS activity (in nmol UDP produced/ min / mg protein) is given by:
D (Es2-Es1) – (D Ebk2-Ebk1)/ 6.22 x 1000/100 x 1000/4 x 1/time x 1/protein (mg/ml)
With E is value of OD for the sample and Ebk value OD for the blank
The blank is carried out with the extract that has been heat inactivated for 5 min at 80°C before addition to the mixture.