N-terminal Protein Labeling Kit - Jena Bioscience

Manual
N-terminal Protein Labeling Kit
Universal N-terminal Protein Labeling with Coumarin
Protein Labeling
Cat.-No. Amount
FP-302-COU
For in vitro use only
Quality guaranteed for 12 months
Store at -20°C
Avoid freeze / thaw cycles
for labeling of
5 mg protein
Coumarin-thioester must be stored in the dark
Kit contents
pET Vector (red cap)
5 µg modified pET-His vector
MPAA (white cap)
35 mg (4-carboxylmethyl) thiophenol
Coumarin-Thioester (purple cap)
12 µl 50 mM in DMSO
TEV protease (N’6His-modified)
230 µg
Description
Coumarin Protein-N-labeling Kit provides a highly
efficient and easy-to-handle tool for the N-terminal
modification of proteins based on an intermolecular
transthioesterification reaction. For N-terminal
labeling, a protein containing an N-terminal cysteine
undergoes a native chemical ligation with thioester
probes.
The kit contains Coumarin-Thioester as fluorescent
label.
Background
Site-specific protein modification can facilitate the
characterization of proteins with respect to their
structure, folding, and interaction with other proteins
both in biochemical and in cellular investigations.
Native chemical ligation has been widely used in
protein synthesis and engineering. The approach is
based on the chemoselective reaction between a
peptide containing a C-terminal thioester and another
peptide containing an N-terminal cysteine. The initial
chemoselective transthioesterification in NCL is
essentially reversible, whereas the subsequent S→N
acyl shift is spontaneous and irreversible. Thus, the
reaction is driven to form an amide bond specifically
at the ligation site, even in the presence of
unprotected internal cysteine residues.
Herein, a method for N-terminal labeling of proteins is
described. A protein containing an N-terminal
cysteine can undergo NCL with thioester probes. The
exposure of an N-terminal cysteine can be achieved
via TEV (tobacco etch virus) protease cleavage.
Structure
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
HS
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OH
MPAA (4-carboxylmethyl)
thiophenol
Spectroscopic data of coumarin
Excitation maximum: λEx = 440 nm
Emission maximum: λEm = 475 nm
Coumarin-Thioester

Manual
N-terminal Protein Labeling Kit
Universal N-terminal Protein Labeling with Coumarin
Protein Labeling
Procedure
Preparation of proteins with N-terminal cysteine
1. Clone target gene containing N-terminal tobacco
etch virus (TEV) protease recognition site
(ENLYFQC) into modified pET-His vector using
NdeI and XhoI sites.
2. Express protein (His-TEV-Cys-protein) in BL21(DE3)
cells.
3. Collect cells in 25 mL ice-cold Breaking Buffer
freshly supplemented with 1 mM PMSF and 2
mM β-mercaptoethanol. CRITICAL: PMSF and
β-mercaptoenthanol should be added freshly.
Don’t add any reducing substances.
4. Lyse cells using a microfluidizer or ultrasonication.
5. Centrifuge cell lysate at 35,000 rpm, 4°C for 30
min.
6. Filter supernatant through a 0.2 µm filter.
7. Load cell lysate onto a Ni-NTA column
equilibrated with Buffer A.
8. Wash column with Buffer A and continue with
2% Buffer B until absorbance reaches baseline.
9. Elute column with a gradient of 2-100% Buffer
B. Collect eluted fractions.
10. Identify and collect fractions of interest by SDS-
PAGE.
11. Add tobacco etch virus (TEV) protease at 1:20
molar ratio to the protein in the dialysis membrane
tubing.
12. Dialyze sample against Dialysis Buffer
overnight.
13. Centrifuge dialyzed solution at 8000 rpm for 10
min. Collect supernatant.
14. Add MgCl2 and imidazole to the supernatant to
a final concentration of 5 mM and 10 mM,
respectively.
15. Load onto a Ni-NTA column equilibrated with
Buffer A. Collect flow-through.
16. Wash column with 2% Buffer B and collect flowthrough.
17. Pool and concentrate protein from flow-through.
18. Run a gel filtration on a Superdex column using
Dialysis Buffer. CRITICAL: Prepare fresh
solution, filter buffer through a 0.2 µm filter and
degas on a vacuum-membrane pump by stirring
for 0.5 h at room temperature.
19. Identify and collect fractions of interest by SDS-
PAGE. Concentrate protein and snap freeze in
liquid nitrogen. Store protein at -80°C.
Protein N-terminal Labeling
20. Incubate 50 µM N-Cys-protein with 500 µM
Coumarin-Thioester in the presence of 200
mM MPAA in Reaction Buffer on ice
overnight. The reaction is monitored by ESI-MS.
21. Remove excess dye using a desalting column preequilibrated
in Dialysis Buffer.
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com Last update: Dec. 22, 2011

Manual
N-terminal Protein Labeling Kit
Universal N-terminal Protein Labeling with Coumarin
Protein Labeling
To be provided
Breaking Buffer
25 mM NaH2PO4 pH 7.5, 0.5 M NaCl
PMSF
β-mercaptoethanol
Buffer A
50 mM NaH2PO4 pH 8.0, 0.3 M NaCl,
2 mM β-mercaptoethanol
Buffer B
50 mM NaH2PO4 pH 8.0, 0.3 M NaCl,
2 mM β-mercaptoethanol, 0.5 M imidazole
Reaction Buffer
30 mM NaH2PO4 pH 7.5, 50 mM NaCl
Dialysis Buffer
30 mM NaH2PO4 pH 7.5, 50 mM NaCl, 2 mM DTE
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com Last update: Dec. 22, 2011

Manual
N-terminal Protein Labeling Kit
Universal N-terminal Protein Labeling with Coumarin
Protein Labeling
Selected References
Yi, L. et al. (2010) A Highly Efficient Strategy for Modification of
Proteins at the C Terminus. Angewandte Chemie Int. Ed. 49:9417.
Yi, L. et al. (2011) One-Pot Dual-Labeling of a Protein via Two
Chemoselective Reactions. Angewandte Chemie Int. Ed. 50: 8287.
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com Last update: Dec. 22, 2011