Site-specific functionalization of nanobodies: From labeling to cellular uptake

Our long-term goal is to develop and validate novel methods to visualize and manipulate biochemical processes within living cells with fluorescently labeled proteins functionalized for cellular uptake and photoinducible activation. We will utilize artificial cell penetrating peptides and optimize them for intracellular release and bioavailability of cargo proteins. For intracellular binding of target structures we will generate specific nanobodies against the nuclear proteins PCNA and Dnmt1. Different strategies for site-selective modification of proteins including intein expression, incorporation of artificial amino acids by amber suppression and bioorthogonal reactions will be employed to introduce functional modules like fluorescent labels, caged amino acids and artificial cell penetrating groups.

This functionalization will initially be tested with GFP to evaluate the conjugation of cyclic peptides and will be optimized for enhanced cellular uptake. We will monitor the uptake of modified nanobodies by live cell microscopy and will activate them with short light pulses. Physiological effects on target structures including the disruption of protein interactions will be quantified by automated image analysis. This proposed collaboration, combines expertise in nanobody engineering, bioorthogonal chemistry, cellular uptake mechanisms, microscopy and cell biology. Consequently, this joint effort should lead to the development of entirely new research tools to detect, study and manipulate antigens in living cells.

Prof. Dr. M. Cristina Cardoso
Technische Universität Darmstadt

Tel.: +49 6151 16 - 2377
Fax: +49 6151 16 - 2375

Email Prof. Cardoso

Prof. Dr. Christian Hackenberger
Humboldt Universität zu Berlin
Leibniz-Institut für Molekulare Pharmakologie (FMP)

Tel.: +49 30 94793 - 181
Fax: +49 30 94793 - 188

Email Prof. Hackenberger

Prof. Dr. Heinrich Leonhardt
LMU München

Tel.: +49 89 2180 - 74232
Fax: +49 89 2180 - 74236

Email Prof. Leonhardt


Publications within the SPP 1623 project

H.Y. Chiu, J.A. Bates, J. Helma, H. Engelke, H. Harz, T. Bein, H. Leonhardt
2018, Epub ahead of print
Nanoparticle Mediated Delivery and Small Molecule Triggered Activation of Proteins in the Nucleus.
Link to the article

D. Schumacher, J. Helma, A.F.L. Schneider, H. Leonhardt, C.P.R. Hackenberger
Angewandte Chem.
2018, 130, 2336-2357
Nanobodys: Strategien zur chemischen Funktionalisierung und intrazelluläre Anwendungen.
Angewandte Chem. Int. Ed. 2018, 57(9), 2314-2333.
Chemical functionalization strategies and intracellular applications of nanobodies.
Link to the article

A.F.L. Schneider, C.P.R. Hackenberger
Curr. Opin. in Biotechnology
2017, 48, 61-68
Flourescent labeling in living cells.
Link to the article

H.D. Herce, D. Schumacher, A.F.L. Schneider, A.K. Ludwig, F.A. Mann, M. Filies, M.-A. Kasper, S. Reinke, E. Krause, H. Leonhardt, M.C. Cardoso, C.P.R. Hackenberger
Nature Chemistry
2017, 9, 762-771
Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.
Link to the article

Link to supplementary movie

D. Schumacher, O. Lemke, J. Helma, L. Gerszonowicz, V. Waller, T. Stoschek, P.M. Durkin, N. Budisa, H. Leonhardt, B.G. Keler, C.P.R. Hackenberger
Chem. Sci.
2017, 8, 3471-3478
Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling
Link to the article

A. Stengl, D. Hörl, H. Leonhardt, J. Helma
SLAS Discovery
2017, 22(3), 309-315
A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies
Link to the article

R. Röder, J. Helma, T. Preiß, J.O. Rädler, H. Leonhardt, E. Wagner
Pharm. Res.
2017, 34, 161-174
Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells
Link to the article

J. Bertran-Vicente, M. Penkert, O. Nieto-Garcia, J.-M. Jeckelmann, P. Schmieder, E. Krause, C.P.R. Hackenberger
Nature Comm.
2016, 7, Article number: 12703
Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides.
Link to the article

S. Schwagerus, O. Reimann, C. Despres, C. Smet-Nocca, C.P.R. Hackenberger
J. Pept. Sci.
2016,22(5), 327-333
Semi-synthesis of a tag-free O-GlcNAcylated tau protein by sequential chemoselective ligation
Link to the article

H.-Y. Chiu, W. Deng, H. Engelke, J. Helma, H. Leonhardt, T. Bein
Sci. Rep.
2016, 6, 25019
Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time.
link to the article

D. Schumacher, C.P.R. Hackenberger, H. Leonhardt, J. Helma
J. Clin. Immunol.
2016, 36, 100-107 (Review)
Current Status: Site-Specific Antobody Drug Conjugates.
Link to the article

E. Hoffmann, K. Streichert, N. Nischan, C. Seitz, T. Brunner, S. Schwagerus, C.P.R. Hackenberger, M. Rubini
Molecular BioSystems
2016, 12, 1750-1755
Stabilization of bacterially expressed erytropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylaton sites.
Link to the article

D. Schumacher, J. Helma, F.A. Mann, G. Pichler, F. Natale, E. Krause, M.C. Cardoso, C.P.R. Hackenberger, H. Leonhardt
Angew. Chem. Int. Ed.
2015, 54(46), 13787-13791
Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase
Link to the article

M. Rajan, O. Mortusewicz, U. Rothbauer, F.D. Hastert, K. Schmidthals, A. Rapp, H. Leonhardt
FEBS Open Bio.
2015, 5, 779 - 788
Generation of an alpaca-derived nanobody recognizing y-H2AX.
Link to the article

K. Klamecka, P.M. Severin, L.F. Milles, H.E. Glaub, H. Leonhardt
Phys. Biol.
2015, 12, 056009
Energy profile of nanobody-GFP complex under force.
Link to the article

J. Helma, M.C. Cardoso, S. Muyldermans, H. Leonhardt
J. Cell. Biol.
2015, 209, 633 - 644.
Nanobodies and recombinant binders in cell biology.
Link to the article

M. Mühlberg, M.G. Hösl, C. Kühne, J. Dernedde, N. Budisa, C.P.R. Hackenberger
Beilstein JOC
2015, 11, 784-791
Orthogonal Dual-Modification of Proteins for the Engineering of Multivalent Protein Scaffolds
Link to the article

C. Witte, V. Martos, H.M. Rose, R. Reinke, S. Klippel, L. Schröder, C.P.R. Hackenberger
Angew. Chem. Int. Ed.
2015, 54(9), 2806-2810 (VIP-Paper)
Live-cell MRI of xenon Hyper-CEST biosensors targeted in metabolically-labeled cell-surface glycans
Link to the article

N. Nischan, H.D. Herce, F. Natale, N. Bohlke, N. Budisa, M.C. Cardoso, C.P.R. Hackenberger
Angew. Chem. Int. Ed.
2015, 54(6), 1950-1953
Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability
Link to the article

O. Reimann, C. Smet-Nocca, C.P.R. Hackenberger
. Chem. Int. Ed. 2015, 54, 306-310
Traceless Purification and Desulfurization of Tau Protein Ligation Products
Link to the article

M.R.J. Vallée, P. Majkut, D. Krause, M. Gerrits, C.P.R. Hackenberger
Chem. Eur. J.
2015, 21, 970-974
Chemoselective Bioconjugation of Triazole Phosphonites in Aqueous Media
Link to the article

D. Aschenbrenner, D.A. Pippig, K. Klamecka, K. Limmer, H. Leonhardt, H.E. Gaub
PLoS One 2014, e115049
Parallel force assay for protein-protein interactions.
Link to the article

H.D. Herce, A.E. Garcia, M.C. Cardoso
J. Am. Chem. Soc.
2014, 136, 17459-17467
Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules
Link to the article

R. Wieneke, N. Labòria, M. Rajan, A. Kollmannsperger, F. Natale, M.C. Cardoso, R. Tampé
J. Am. Chem. Soc.
2014, 136, 13975-13978
Live-cell Targeting of His-tagged Proteins by Multivalent N-Nitrilotriacetic Acid Carrier Complexe
Link to the article

D. Schumacher, C.P.R. Hackenberger
Curr. Opin. Chem. Biol.
2014, 22, 62-69
More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins
Link to the article

J. Bertran-Vicente, R.A. Serwa, M. Schümann, P. Schmieder, E. Krause, C.P.R. Hackenberger
J. Am Chem. Soc.
2014, 136(39), 13622-13628
Site-Specifically Phosphorylated Lysine Peptides
Link to the article

P. Majkut, I. Claußnitzer, H. Merk, C. Freund, C.P.R. Hackenberger, M. Gerrits
2013, 8(12): e82352
Completion of Proteomic Data Sets by Kd Measurement Using Cell-Free Synthesis
of Site-Specifically Labeled Proteins
Link zum Artikel

H.D. Herce, W. Deng, J. Helma, H. Leonhardt, M.C. Cardoso
Nat. Commun 2013, 4, 2660
Visualization and targeted disruption of protein interactions in living cells.
Link to the article

N. Nischan, A. Chakrabarti, R.A. Serwa, P.H.M. Bovee-Geurts, R. Brock, C.P.R. Hackenberger
Angew. Chem.
2013, 125, 45, 12138-12142
Stabilisierung von Peptiden für intrazelluläre Anwendungen mit Phosphoramidat-verzweigten Polyethylenglycol-Ketten
Angew. Chem. Int. Ed. 2013, 52, 45, 11920-11924
Stabilization of Peptides for Intracellular Applications by Phosphoramidate-Linked PEG Chains
Link zum Artikel

M.R.J. Vallée, L.M. Artner, J. Dernedde, C.P.R. Hackenberger
Angew. Chem.
2013, 125, 9682-9686
Angew. Chem. Int. Ed.
2013, 52, 9504-9508
Alkyne phophonites for sequential azide-azide couplings
Link zum Artikel