Spot-Tag: a Nanobody-based Peptide-Tag System for Protein Detection, Puri� cation and Imaging
Michael Metterlein1, Larisa Yurlova1, Andrea Buchfellner1, Benjamin Ruf1, Verena Leingärtner1, Jacqueline Bogner1, Tina Romer1, Philipp D. Kaiser2, Felix Hartlepp1, Christian Linke-Winnebeck1*
1ChromoTek GmbH, Planegg-Martinsried, Germany, 2Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany*Email to: [email protected]
Acknowledgements:This work was supported by a grant “Zentrales Innovationsprogramm Mittelstand“ (ZIM) from the Federal Ministry for Economic A� airs and Energy of Germany. Also, we thank Andreas Thomae at the Core Facility Bioimaging of the Biomedical Center, LMU Munich, for recording confocal and STED images.
References:[1] Tränkle, B. et al.: Monitoring interactions and dynamics of endogenous β-catenin with intracellular nanobodies in living cells. Molecular & Cellular Proteomics 14(3), pp. 707-723 (2015).[2] Braun, M.B. et al.: Peptides in headlock - a novel high-a� nity and versatile peptide-binding nanobody for proteomics and microscopy. Scienti� c reports 6, 19211 (2016).[3] Virant, D. et al.: A peptide tag-speci� c nanobody enables high-quality labeling for dSTORM imaging. Nature Communications 9(1) 930 (2018).
Disclaimer:For research use only. ChromoTek`s products and technology are covered by granted patents and pending applications owned by or available to ChromoTek GmbH by exclusive license. ChromoTek, Chromobody, Spot-Tag and Spot-Trap are registered trademarks of ChromoTek GmbH. Nanobody is a registered trademark of Ablynx nv.
We have developed a new epitope tag system based on a VHH (also called nanobody®), i.e. a small and highly stable alpaca single-domain antibody. This VHH binds with high a� nity to the Spot-Tag®, an engineered 12-amino acid sequence (PDRVRAVSHWSS) derived from a linear epitope within β-catenin. Owing to the unique properties of the anti-Spot-Tag-VHH, the Spot-Tag capture and detection system is universally applicable.
The Spot-Tag
The anti-Spot nanobody
Applications of the Spot-Tag system
Immunisation of alpacas with β-catenin yields nanobody that binds unstructured N-terminus[1] (amino acids 16-27).
Further screening of 30 variants identi� es tag sequence optimised for high a� nity: the Spot-Tag.
100 200 300 4000.0
0.2
0.4
0.6
0.8
BC2-mCherry
Time (s)
Bind
ing
(nm
)
5 nM10 nM15 nM25 nM50 nM
100 200 300 4000.0
0.2
0.4
0.6
0.8
Spot-mCherry
Time (s)
Bind
ing
(nm
)
5 nM10 nM15 nM25 nM50 nM
Origin of the Spot-Tag
High a� nity of the Spot-Tag→ Engineering of original epitope leads to signi� cantly decreased dissociation and thus improved a� nity for Spot-Tag.
→ High a� nity binding of Spot-Tag to anti-Spot nanobodyKD kon (M
-1s-1) ko� (s-1)
β-cat. 16-27 C-term. 45 nM 1.5 ×105 6.7 ×10-3
β-cat. 16-27 N-term. 56 nM 1.4 ×105 8.0 ×10-3
Spot-Tag C-term. 7 nM 1.8 ×105 1.3 ×10-3
Spot-Tag N-term. 6 nM 1.3 ×105 7.4 ×10-4
β-cat.-mCherry Spot-Tag-mCherry
0 2 4 6 8
1.20
1.25
1.30
Urea [M]
ratio
F(35
0nm
/330
nm)
20 40 60 801.10
1.15
1.20
1.25
Temperature (°C)
Ratio
F(35
0nm
/330
nm)
20 40 60 80
-0.0006
-0.0004
-0.0002
0.0000
0.0002
Temperature (°C)
Firs
tder
ivat
ive
20 40 60 80
0.05
0.10
0.15
Temperature (°C)
Scat
terin
g(A
U)
Crystal structure of the anti-Spot nanobody.[2]
Characteristics
High stability
Firs
t der
ivat
ive
• Single peptide of 131 amino acids• Small size: 14.7 kDa• Binds Spot-Tag with high a� nity• Thorough biochemical and structural characterisation• Recombinant production and thus high batch-to-batch consistency• Binds Spot-Tag fused to N- or C-terminus of a protein• Easily derivatised using � uorophores or agarose matrices
Western blotELISA
Immuno� uorescence microscopy using Spot-Label
Puri� cation and immunoprecipitation using Spot-Trap®
A bivalent format of anti-Spot nanobody conjugated to � uorophores (called Spot-Label), allows the imaging of cellular proteins and structures using � uorescence microscopy.
→ Small size of Spot-Label leads to better tissue penetration.
→ Spot-Label is the first detection tool directed against a small peptide tag that is applicable to super resolution microscopy studies (STED and also STORM[3]) owing to minimal label displacement.
Spot-Label ATTO594 confocal microscopy imaging of Tom70-GFP-Spot in HeLa cells.
Spot-Label ATTO594 STED super resolution imaging of Vimentin-Spot in HeLa cells.
GFP-Spot in HEK293T cell lysate0 50 25 12 6 3ng:
25 kDa
32 kDa
80 kDa
11 kDa
Western blot detection using monovalent Spot-Label ATTO488.
For Western blot detection of Spot-Tag proteins, monovalent Spot-Label conjugated to a � uorophore or in conjuction with a secondary antibody can be used.
→ Spot-Label ATTO488 allows sensitive one-step Western blot detection with minimal background.
→ Alternatively, anti-Spot nanobody can be detected using anti-llama HPR antibody.
-9 -8 -7 -6 -5
0.0
0.5
1.0
M
Abso
rptio
n45
0nm
mCherry controlmCherry-Spot
→ Anti-Spot nanobody allows detection and capture of Spot-Tag proteins in ELISA.
Coating of mCherry-Spot followed by ELISA analysis using varyiing concentrations of anti-Spot nanobody and anti-llama-HRP.
Di� erential scanning � uorimetry analysis of anti-Spot nanobody.
No aggregation observed for anti-Spot nanobody (1 g/l) over a temperature gradient.
Chemical stability of anti-Spot nanobody in a chaotrope (urea).
The anti-Spot nanobody
→ has a melting temperature Tm of 63 °C.
→ shows unusually high colloidal stability.
→ is highly resistant to chaotropes.
Spot-Label ATTO594 STED super resolution imaging of Actin-Chromobody-Spot in HeLa cells.
Anti-Spot nanobody covalently coupled to agarose beads or magnetic agarose beads (called Spot-Trap) enables the immunoprecipitation and puri� cation of Spot-Tag fusion proteins.
→ High a� nity allows the puri� cation of low-abundance proteins.
→ Bound protein can be eluted using free Spot peptide.
→ High chemical stability leads to extraordinary chemical compatibility of Spot-Trap
→ Chemical stability also permits repeated use of Spot-Trap
Input
Flow-
thro
ugh
Elutio
n
80 kDa
25 kDa
32 kDa
11 kDa
Input
Flow-
thro
ugh
Elution fractions
80 kDa
25 kDa
32 kDa
11 kDa
Spot-Trap immunopre-cipitation of GFP-Spot from HEK 293T lysate.
Spot-Trap a� nity puri� cation of GFP-Spot from HEK 293T lysate followed by step-wise Spot peptide (100 µM) elution.
1 M N
aCl
2 M N
aCl
0.5 %
IGEP
AL CA
-630
1 % IG
EPAL
CA-63
0
2 % IG
EPAL
CA-63
0
0.1 %
SDS
0.1 %
Trito
n X-10
0
0.5 %
Trito
n X-10
0
1 % Tr
iton X
-100
1 M ur
ea
2 M ur
ea
4 M ur
ea
6 M ur
ea
8 M ur
ea
0.2 %
SDS
Cont
rol
Cont
rol
1 mM
DTT
5 mM
DTT
10 m
M DT
T
mCherry-Spot
Bu� er compatibility test for the Spot-Trap. After precipitation of mCherry-Spot from E. coli, Spot-Trap was washed using control bu� er or bu� er supplemented with indicated additives.
Glut
athion
e cell
ulose
(sup
pl. 1)
α-c-M
yc 9E
1 (su
pplie
r 1)
α-c-M
yc 9E
1 (su
pplie
r 2)
α-FL
AG (s
uppli
er 3)
Dyna
bead
s (su
pplie
r 2)
Spot
-Trap
(mag
n. ag
arose
)
Spot
-Trap
(aga
rose
)
80 kDa
25 kDa
32 kDa
11 kDa
Background of di� erent a� nity media in immunoprecipitation from HEK 293T lysate without cognate antigen present.
Input Flow-through 1-5 Elution 1-5
Test of long-term stability of Spot-Trap. Spot-Trap was subjected to � ve full cycles of a� nity puri� cation (loading, washing, peptide elution, regeneration) of mCherry-Spot from E. coli. Analysis using Western blotting.
ConclusionThe Spot-Tag system combines the high a� nity and speci� city of an antibody-epitope tag system with the stability and small size of an alpaca nanobody. This results in a universal tag-system that will simplify the puri� cation and concurrent analysis of target proteins.
Nanobody
NC
β-catenin
aa16-27Invariable positions (tag:nanobody interactions)
Screened and mutated positions
Spot-Tag: PDRVRAVSHWSS
Conventional antibody Alpaca heavy chain antibody
Nanobody/VHH
VH
VLCL
CH1
CH2
CH3
VHH
Spot-Trap®Purification
Immunoprecipitation
Spot-Label, bivalentFluorescence microscopy
Super resolution microscopy
Spot-Label, monov.Western blot
anti-Spot VHHWestern blot
ELISA
Alpaca
Biolayer interferometry (BLI) kinetics analysis of binding of Spot-Tag nanobody to mCherry tagged with β-catenin 16-27 or Spot-Tag.
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