Application Note enzymatic activity assay

One step purification of GFP-tagged enzymes for enzymatic
activity test

Background
Expression of fluorescent fusion proteins in cells is a common method. However, a critical question iswhether the fluorescent fusion proteins expressed in cells are active and have properties similar to theirendogenous counterparts. Using the GFP-Trap®, fluorescent fusion proteins can be efficiently pulled out from
cellular extracts for further evaluation in biochemical studies. DNA methyltransferase 1 (DNMT1) is the mostabundant DNA methyltransferase in mammalian cells. In our experiment we analyzed the enzymatic activityof GFP-DNA methyltransferase 1 (DNMT1) bound to GFP-Trap® beads and compared the activity of thefluorescent fusion protein to an enzymatically inactive version of DNMT1 and recombinant DNMT1 (1).

Materials and Methods
GFP-DNMT1 or GFP-DNMT1 (P1229W), a catalytically inactive version of DNMT1, was expressed inHEK293T cells. GFP fusion proteins were immunoprecipitated from cell extracts using the GFP-Trap® asdescribed (1). After pull down, GFP-Trap® A beads binding GFP-DNMT1 were washed extensively in dilution
buffer containing 300 mM NaCl and afterwards twice in 500 ml assay buffer (100 mM KCl, 10 mM Tris pH
7,6, 1 mM EDTA, 1 mM DTT). Amount of immunoprecipitated GFP-DNMT1 bound to GFP-Trap® beads wereanalysed by SDS-PAGE followed by Coomassie staining. 30 µl of methylation mixture (0,1 mCi of S(3H)adensoylmethionine (Amersham Biosciences, 1.67 pmol/ml hemimethylated double-stranded 35-bp DNA(50 pmol/ml), 160 ng/ml BSA) were added to 4 µg GFP-DNMT1 bound to beads. As a positive control 2 µg ofrecombinant purified DNMT1 was used. Incubation was carried out for 2,5 h at 37 °C, and the reactions werespotted onto DE81 cellulose filters. Subsequently, filters were washed three times with 0,2 M (NH4)HCO3,once with 100% EtOH. After drying at 80°C the filter was transferred into a Mini-Poly-Q vial with 5 ml of
Ultima Gold LSC mixture (PerkinElmer Life Sciences) and each sample was measured for 1 min in ascintillation counter (Beckman LS1801). A sample without enzyme was used as negative control.

Application Note enzymatic activity assay

1Version 2011-11-30

Application Note enzymatic activity assay

Results
Both GFP-DNMT1 variants were efficiently pulled
out from cellular extracts. In an enzymatic activity
test

GFP-DNMT1

methyltransferase

showedactivity

aspecificDNA

similartopurifiedrecombinant DNMT1, proving that GFP fusion
proteins

retaintheir

enzymatic

purification with the GFP-Trap®

activity

after

while bound tobeads. In comparison no enzymatic activity wasmeasured with the catalytically inactive DNMT1variant, revealing that endogenous DNMT1 activities

Fig. 1 Soluble protein extracts containing GFP-DNMT1 or
GFP-DNMT1 (P1229W) were one step purified with GFPTrap® beads and analyzed by SDS- Page followed by
Coomassie Blue staining (Top). DNMT1 activity of purified
GFP fusion proteins (∼ 4µg each) and recombinant DNMT1(∼ 2 µg each) were determined (bottom). n=4; error bars
show standard deviationof mammalian cells were efficiently removed by thisquick one step purification (Fig1).

Conclusion
In summary, we showed that the GFP-Trap® is a powerful tool for quick and reliable biochemical
characterization of fluorescent proteins. Enzymatically active fluorescent fusion proteins can be efficiently
isolated with the GFP-Trap® and enzymatic assays are feasible without further purification or elution of thefluorescent fusion protein from beads.

References/relevant literature(1) Rothbauer, U. et al. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics
7, 282-289 (2008)
Reininger, L., et al. An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum
erythrocytic schizogony. Mol. Microbiol 79: 205-221 (2011)
Wang, Y., et al. Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation. Mol.
Biol. Cell: 2270-2281 (2011)
Doskocilova A., et al. A nodulin/ glutamine synthetase-like fusion protein is implicated in the regulation of root morpogenesis and in
signalling triggered by flagellin. Planta 234 (3): 459-76 (2011)

Ordering Information
ProductGFP-Trap®_A

Quantity
20 reactions

Codegta-20

GFP-Trap®_A kit

20 reactions

gtak-20

http://www.chromotek.com

Application Note enzymatic activity assay

2

Version 2011-11-30