The
Plückthun
Lab

Main Research Topics

Engineering and Directed Evolution of Proteins

We study the creation of new proteins and protein variants. The purpose of this work is to use such engineered proteins to enable research and applications which have been very difficult or even impossible so far. Examples of our endeavors are the creation of new engineered binding proteins to inhibit other proteins or to kill tumor cells, or the stabilization of proteins so that they can be studied structurally and biophysically.

Our main areas of interest are novel scaffolds for selective binding (e.g. the DARPin technology we have developed), synthetic antibodies and G-protein-coupled receptors evolved to high stability and expression levels.

Because of the complexity of these tasks, this research requires a highly interdisciplinary approach, combining detailed biophysical studies, computer modeling and advanced molecular biology, especially directed evolution.

Many projects have close collaboration with crystallography, NMR or computational biology. Some projects also bridge protein engineering with applications, in cell biology or, in the case of tumor targeting testing in animal models.


Designed Ankyrin Repeat Proteins as Novel Scaffolds of Selective Binding

AnkyrinOver the last few years, we have developed designed repeat proteins as an alternative to antibodies. By using consensus engineering, libraries of repeat proteins have been designed from which specific, high-affinity binding proteins can be selected. Most of our work so far has concentrated on Designed Ankyrin Repeat Proteins (DARPins). Why are they so useful? They are very stable and do not have disulfide bonds and therefore, unlike most antibodies, also work inside the cell. They can be prepared in very large amounts from E. coli, and show affinities up to the picomolar range. What can you do with them? They are being developed in the research group as tumor targeting reagents, as intracellular inhibitors to study and influence signaling inside the cell, for co-crystallization of membrane proteins, and as model proteins to study fundamental questions of protein folding and design.

See our publications on repeat proteins.


Designed Armadillo Repeat Proteins for modular binding to Peptides

Armadillo repeat proteinOne of the fundamental challenges in future proteomics research will be to generate specific recognition reagents rapidly, and for an ever increasing number of proteomes. One potential solution to this problem would be a modular recognition of peptides, amino acid by amino acid, from pre-selected modules. This is somewhat analogous to the recognition of one strand of DNA by the other. The approach taken is the design and engineering of synthetic Armadillo Repeat Proteins, which have the ability to bind linear, stretched-out peptides, to ultimately create sequence-specific binding. Using a combination of design, directed evolution and structure determinations by crystallography and NMR, this challenging project is bringing together all methods of protein engineering.

See our publication on armadillo repeat proteins..


Stability Engineering of Membrane Proteins, especially G-protein coupled receptors

membrane proteinOne of the biggest challenges in the study of the structure and biophysics of membrane proteins is that these proteins are extremely difficult to handle: They are hard to produce in large amounts, they are very unstable and they often denature when solubilized in detergent. This is especially true for eukaryotic membrane proteins, such as G-protein-coupled receptors (GPCRs), which are the targets of about half of all pharmaceuticals in use today. We recently developed a technology with which GPCRs can be evolved to higher expression, using an E. coli FACS selection strategy. The evolved proteins express at much higher levels not only in bacteria, but also in Pichia pastoris and mammalian cells. Moreover, the detergent-solubilized proteins are clearly more stable against denaturation — an important prerequisite for structure determination. This work should not only make more eukaryotic membrane proteins amenable to study, but help elucidate the structural rules of stable membrane proteins. We also developed strategies to select binding proteins (e.g. Designed Ankyrin Repeat Proteins or antibody fragments) against the native state of integral membrane proteins and use such proteins to support stabilization and crystallization.

See our publications on membrane proteins.


Antibody engineering

antibodyOver the years, we investigated and engineered stable antibody frameworks with significantly better properties than natural ones. This work lead to such a stability-engineered antibody against EpCAM being now in clinical trials. Also, extremely tight binders were evolved in vitro against proteins, peptides and small molecules, up to 1 pM (a monovalent scFv against the prion protein). It is usually impossible to obtain such high affinities from immunized animals, but it is now possible from libraries, using directed evolution. Also, libraries can be created that encode proteins with superior physical properties. Recent questions investigated include whether the same structural features play a role when the proteins are made in different formats and in prokaryotic or eukaryotic cells, and what features prevent misfolding and aggregation. Clearly, the answers to these questions not only are of fundamental interest, but also have immediate applications in the many uses of antibodies.

See our publications on antibody engineering.


Protein Engineering Applied to Tumor Targeting

tumor targetingOne of the goals of the engineering efforts in the lab is the design of superior proteins for therapy, predominantly for tumor targeting. Both recombinant antibodies and Designed Ankyrin Repeat Proteins (DARPins) are being used for this purpose. The focus is on fundamental studies, to understand the interdependencies of affinity, valency, size, stability and effector engineering on in vivo efficacy. Novel molecular formats are being developed, combining a targeting function with an effector function. This work combines meticulous biophysical characterization of new proteins, tests in cell culture and measuring biodistribution and efficacy in animal models. The goal of this work is to engineer targeting proteins rationally, with a clear understanding of the desired properties. Having access to state-of-the-art modeling and directed evolution, we are not limited to simply making fusion proteins from available natural components but can significantly improve the properties of the constituent modules.

See our publications on tumor targeting.


Technologies used: New Strategies of Directed Evolution

technologiesMany of the studies in the various projects required technologies that were simply not available at the time. Therefore, we have always been developing novel technologies to be able to greatly expand our toolbox, in order to make certain developments in molecular engineering possible in the first place. One example is ribosome display, a method of cell-free selection and evolution from very large libraries that is now widely used. It has been adapted to affinity maturation (the current record is a monovalent dissociation constant of 1 pM), protein stability, or enzymatic turnover. Another example is phage display, which has recently been adapted to make it compatible with a wider spectrum of proteins to be displayed (by changing the export pathway), and has also been used to select for properties other than mere binding, such as protein stability and enzymatic turnover. A third example is the evolution of stable membrane proteins by using FACS, described above. These methods are not developed for their own sake but driven by the necessity to solve a protein engineering problem.

Relevant Publications