Spot-Tag: a Nanobody-based Peptide-Tag System for Protein ......Absorption 450 nm mCherry control...
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Spot-Tag: a Nanobody-based Peptide-Tag System for Protein Detection, Purification and Imaging Michael Metterlein 1 , Larisa Yurlova 1 , Andrea Buchfellner 1 , Benjamin Ruf 1 , Verena Leingärtner 1 , Jacqueline Bogner 1 , Tina Romer 1 , Philipp D. Kaiser 2 , Felix Hartlepp 1 , Christian Linke-Winnebeck 1* 1 ChromoTek GmbH, Planegg-Martinsried, Germany, 2 Natural 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 Affairs 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-affinity and versatile peptide-binding nanobody for proteomics and microscopy. Scientific reports 6, 19211 (2016). [3] Virant, D. et al.: A peptide tag-specific 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 V H H (also called nanobody®), i.e. a small and highly stable alpaca single-domain antibody. This V H H binds with high affinity 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-V H H, 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 identifies tag sequence optimised for high affinity: the Spot-Tag. 100 200 300 400 0.0 0.2 0.4 0.6 0.8 Time (s) Binding (nm) 100 200 300 400 0.0 0.2 0.4 0.6 0.8 Time (s) 5 nM 10 nM 15 nM 25 nM 50 nM Origin of the Spot-Tag High affinity of the Spot-Tag → Engineering of original epitope leads to significantly decreased dissociation and thus improved affinity for Spot-Tag. → High affinity binding of Spot-Tag to anti-Spot nanobody K D k on (M -1 s -1 ) k off (s -1 ) β-cat. 16-27 C-term. 45 nM 1.5 ×10 5 6.7 ×10 -3 β-cat. 16-27 N-term. 56 nM 1.4 ×10 5 8.0 ×10 -3 Spot-Tag C-term. 7 nM 1.8 ×10 5 1.3 ×10 -3 Spot-Tag N-term. 6 nM 1.3 ×10 5 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(350nm/330nm) 20 40 60 80 1.10 1.15 1.20 1.25 Temperature (°C) Ratio F(350 nm/330 nm) 20 40 60 80 Temperature (°C) 20 40 60 80 0.05 0.10 0.15 Temperature (°C) Scattering (AU) Crystal structure of the anti-Spot nanobody. [2] Characteristics High stability First derivative • Single peptide of 131 amino acids • Small size: 14.7 kDa • Binds Spot-Tag with high affinity • 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 fluorophores or agarose matrices Western blot ELISA Immunofluorescence microscopy using Spot-Label Purification and immunoprecipitation using Spot-Trap® A bivalent format of anti-Spot nanobody conjugated to fluorophores (called Spot-Label), allows the imaging of cellular proteins and structures using fluorescence 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 lysate 0 50 25 12 6 3 ng: 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 fluorophore 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 Absorption 450 nm mCherry control mCherry-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. Differential scanning fluorimetry 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 T m 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 purification of Spot-Tag fusion proteins. → High affinity allows the purification 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-through Elution 80 kDa 25 kDa 32 kDa 11 kDa Input Flow-through Elution fractions 80 kDa 25 kDa 32 kDa 11 kDa Spot-Trap immunopre- cipitation of GFP-Spot from HEK 293T lysate. Spot-Trap affinity purification of GFP-Spot from HEK 293T lysate followed by step-wise Spot peptide (100 μM) elution. 1 M NaCl 2 M NaCl 0.5 % IGEPAL CA-630 1 % IGEPAL CA-630 2 % IGEPAL CA-630 0.1 % SDS 0.1 % Triton X-100 0.5 % Triton X-100 1 % Triton X-100 1 M urea 2 M urea 4 M urea 6 M urea 8 M urea 0.2 % SDS Control Control 1 mM DTT 5 mM DTT 10 mM DTT mCherry-Spot Buffer compatibility test for the Spot-Trap. After precipitation of mCherry-Spot from E. coli, Spot-Trap was washed using control buffer or buffer supplemented with indicated additives. Glutathione cellulose (suppl. 1) α-c-Myc 9E1 (supplier 1) α-c-Myc 9E1 (supplier 2) α-FLAG (supplier 3) Dynabeads (supplier 2) Spot-Trap (magn. agarose) Spot-Trap (agarose) 80 kDa 25 kDa 32 kDa 11 kDa Background of different affinity 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 five full cycles of affinity purification (loading, washing, peptide elution, regeneration) of mCherry-Spot from E. coli. Analysis using Western blotting. Conclusion The Spot-Tag system combines the high affinity and specificity 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 purification and concurrent analysis of target proteins. Nanobody N C β-catenin aa16-27 Invariable positions (tag:nanobody interactions) Screened and mutated positions Spot-Tag: PD R VR A V S H W SS Conventional antibody Alpaca heavy chain antibody Nanobody/ V H H V H V L C L C H 1 C H 2 C H 3 V H H Spot-Trap® Purification Immunoprecipitation Spot-Label, bivalent Fluorescence microscopy Super resolution microscopy Spot-Label, monov. Western blot anti-Spot V H H Western 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.
Transcript of Spot-Tag: a Nanobody-based Peptide-Tag System for Protein ......Absorption 450 nm mCherry control...
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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.
