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

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 BioSystems2016, 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