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: c.linkewinnebeck@chromotek.com

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.