Protein Engineering

Our long-term research goal is to understand the details of how proteins interact in cellular context and how these interactions can be modulated in a therapeutic setting. To reach these goals, we generate affinity reagents that specifically and with high affinity bind their target molecule (antigen) and thereby influence its function. For the construction of these affinity reagents we apply a structure-guided protein engineering approach and we simultaneously screen 10 billion independent protein variants by phage display for improved properties. These affinity reagents are used in research, diagnosis and therapy of neurodegenerative and inflammatory diseases addressing unmet medical needs.

© Fraunhofer IME, Goethe-University | Andreas Ernst

© Wiechmann, Gärtner et al. 2017 ¹
SUMO2 as grey ribbon. Numbered spheres represent randomized positions in three different regions (orange, purple, turquoise).

Intracellular Inhibitors Engineering

We engineer intracellular affinity reagents/inhibitors based on ubiquitin and ubiquitin-like proteins (UbiL-Ins). Using a structure-guided approach and phage display, we engineer interface residues to optimize the intermolecular contacts to various intracellular target proteins. This process yields highly selective inhibitors with improved affinity and specificity. Thus far, several inhibitors of proteins involved in the ubiquitin-proteasome system (DUBs, E3s and linear binding motifs) have been established (Ernst et al., 2013). In ongoing research projects, we expanded this engineering pipeline to other proteins with a ubiquitin-like fold:

- SUMO1 and 2 involved in DNA damage response and ribosome biogenesis
- LC3B involved in selective and non-specific autophagy pathways
- Ubiquitin-like effector domains targeting small GTPases of the Ras superfamily.

© Fraunhofer IME, Goethe-Universität | Andreas Ernst und Mateusz Putyrski
IgG scheme. Light chain (in blue) and heavy chain (in green).

Antibody Engineering

We constructed our own combinatorial synthetic Fab library containing up to 10 billion different antibody fragments. This library has been successfully used to select high affinity binders, specific for several intra- and extracellular proteins. For example, the selected antibodies neutralize the activity of a novel member of the Interleukin-1 family of cytokines. Our pipeline includes phage selections, bacterial expression of Fab fragments and their characterization as well as production of full length IgGs in mammalian cell culture.

Cytokine Engineering

Cytokines are extracellular mediators, which orchestrate the function of the immune system. Their activity is under tight spatiotemporal control, so that the active molecules exhibit relatively short serum half-life. This, in turn, limits the use of exogenous cytokine preparations as therapeutics. Our efforts aim at engineering cytokines to enhance their stability and biological half-life. In this project, by stabilizing IL-1 members, we attempt to establish novel putative therapeutics to treat autoimmune disorders and cancer.

Han Y, Mora J, Huard A, da Silva P, Wiechmann S, Putyrski M, Schuster C, Elwakeel E, Lang G, Scholz A, Scholz T, Schmid T, de Bruin N, Billuart P, Sala C, Burkhardt H, Parnham MJ, Ernst A, Brüne B, Weigert A. IL-38 Ameliorates Skin Inflammation and Limits IL-17 Production from γδ T Cells. Cell Rep 2019. 27 (3): 835-846.e5.

von Knethen A, Schäfer A, Kuchler L, Knape T, Christen U, Hintermann E, Fißlthaler B, Schröder K, Brandes RP, Genz B, Abshagen K, Pützer BM, Sha LK, Weigert A, Syed SN, Schulz M, Shah AM, Ernst A, Putyrski M, Finkelmeier F, Pesic M, Greten F, Hogardt M, Kempf VAJ, Gunne S, Parnham MJ, Brüne B. Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis. Theranostics 2019. 9 (7): 2003-2016.

Lim R, Sugino T, Nolte H, Andrade J, Zimmermann B, Shi C, Doddaballapur A, Ong YT, Wilhelm K, Fasse JWD, Ernst A, Kaulich M, Husnjak K, Boettger T, Guenther S, Braun T, Krüger M, Benedito R, Dikic I, Potente M. Deubiquitinase USP10 regulates Notch signaling in the endothelium. Science 2019. 364 (6436): 188-193.

Manczyk N, Veggiani G, Gish GD, Yates BP, Ernst A, Sidhu SS, Sicheri F. Dimerization of a ubiquitin variant leads to high affinity interactions with a ubiquitin interacting motif. Protein Sci. 2019. 28 (5): 848-856.

Wegner M, Diehl V, Bittl V, de Bruyn R, Wiechmann S, Matthess Y, Hebel M, Hayes MG, Schaubeck S, Benner C, Heinz S, Bremm A, Dikic I, Ernst A, Kaulich M. Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome. Elife 2019. 8.


Canny, M.D., Moatti, N., Wan, L.C.K., Fradet-Turcotte, A., Krasner, D., Mateos-Gomez, P.A., Zimmermann, M., Orthwein, A., Juang, Y.-C., Zhang, W., Noordermeer, S.M., Seclen, E., Wilson, M.D., Vorobyov, A., Munro, M., Ernst, A., Ng, T.F., Cho, T., Cannon, P.M., Sidhu, S.S., Sicheri, F., Durocher, D. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency (2018) Nature Biotechnology, 36 (1), 95-102.


Manczyk, N., Yates, B.P., Veggiani, G., Ernst, A., Sicheri, F., Sidhu, S.S. Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif (2017) Protein Science, 26 (5), 1060-1069.

Stolz, A., Putyrski, M., Kutle, I., Huber, J., Wang, C., Major, V., Sidhu, S.S., Youle, R.J., Rogov, V.V., Dötsch, V., Ernst, A., Dikic, I. Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins (2017) EMBO Journal, 36 (4), 549-564.

Wiechmann, S., Ernst, A. Engineering of intracellular modulators [Engineering von intrazellulären Modulatoren] (2017) BioSpektrum, 23 (7), 769-771.

Wiechmann, S., Gärtner, A., Kniss, A., Stengl, A., Behrends, C., Rogov, V.V., Rodriguez, M.S., Dötsch, V., Müller, S., Ernst, A. Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation (2017) Journal of Biological Chemistry, 292 (37), 15340-15351.

Zhang, W., Sartori, M.A., Makhnevych, T., Federowicz, K.E., Dong, X., Liu, L., Nim, S., Dong, A., Yang, J., Li, Y., Haddad, D., Ernst, A., Heerding, D., Tong, Y., Moffat, J., Sidhu, S.S. Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10 (2017) Journal of Molecular Biology, 429 (22), 3546-3560.


Mora, J., Schlemmer, A., Wittig, I., Richter, F., Putyrski, M., Frank, A.-C., Han, Y., Jung, M., Ernst, A., Weigert, A., Brüne, B. Interleukin-38 is released from apoptotic cells to limit inflammatory macrophage responses (2016) Journal of Molecular Cell Biology, 8 (5), 426-438.


Ernst, A., Appleton, B.A., Ivarsson, Y., Zhang, Y., Gfeller, D., Wiesmann, C., Sidhu, S.S. A structural portrait of the PDZ domain family (2014) Journal of Molecular Biology, 426 (21), 3509-3519.

Gfeller, D., Ernst, A., Jarvik, N., Sidhu, S.S., Bader, G.D. Prediction and experimental characterization of nsSNPs altering human PDZ-binding motifs (2014) PLoS ONE, 9 (4), art. no. e94507.

Stolz, A., Ernst, A., Dikic, I. Cargo recognition and trafficking in selective autophagy (2014) Nature Cell Biology, 16 (6), 495-501.


Ernst, A., Avvakumov, G., Tong, J., Fan, Y., Zhao, Y., Alberts, P., Persaud, A., Walker, J.R., Neculai, A.-M., Neculai, D., Vorobyov, A., Garg, P., Beatty, L., Chan, P.-K., Juang, Y.-C., Landry, M.-C., Yeh, C., Zeqiraj, E., Karamboulas, K., Allali-Hassani, A., Vedadi, M., Tyers, M., Moffat, J., Sicheri, F., Pelletier, L., Durocher, D., Raught, B., Rotin, D., Yang, J., Moran, M.F., Dhe-Paganon, S., Sidhu, S.S. A strategy for modulation of enzymes in the ubiquitin system (2013) Science, 339 (6119), 590-595.

Ernst, A., Sidhu, S.S. Engineering ubiquitin to modulate the ubiquitin proteosome system (2013) Cell Cycle, 12 (11), 1651-1652.


Gfeller, D., Butty, F., Wierzbicka, M., Verschueren, E., Vanhee, P., Huang, H., Ernst, A., Dar, N., Stagljar, I., Serrano, L., Sidhu, S.S., Bader, G.D., Kim, P.M. The multiple-specificity landscape of modular peptide recognition domains (2011) Molecular Systems Biology, 7, art. no. 484.


Ernst, A., Gfeller, D., Kan, Z., Seshagiri, S., Kim, P.M., Bader, G.D., Sidhu, S.S. Coevolution of PDZ domain-ligand interactions analyzed by high-throughput phage display and deep sequencing (2010) Molecular BioSystems, 6 (10), 1782-1790.


Ernst, A., Sazinsky, S.L., Hui, S., Currell, B., Dharsee, M., Seshagiri, S., Bader, G.D., Sidhu, S.S. Rapid evolution of functional complexity in a domain family (2009) Science Signaling, 2 (87).


Matsuura, T., Ernst, A., Zechel, D.L., Plückthun, A. Combinatorial approaches to novel proteins (2004) ChemBioChem, 5 (2), 177-182.


Matsuura, T., Ernst, A., Plückthun, A. Construction and characterization of protein libraries composed of secondary structure modules (2002) Protein Science, 11 (11), 2631-2643.

¹ This picture was originally published in the Journal of Biological Chemistry. Wiechmann S, Gärtner A, Kniss A, Stengl A, Behrends C, Rogov VV, Rodriguez MS, Dötsch V, Müller S, Ernst A. Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation. J. Biol. Chem. 2017; 292 (37): 15340-15351.