A hyperstable Minimalist Protein for molecular recognition
- Biotechnologie
Abstract
Antibodies represent the most rapidly growing class of therapeutic proteins. They are used for the treatment of a variety of diseases, including various types of cancer and immunological disorders. The most important property of antibodies is their ability to bind to virtually any antigen with high affinity and specificity. Nowadays, display technologies such as ribosome, phage or yeast display enable the development of alternative binding scaffolds, which, like antibodies, have the ability to strongly and specifically bind to any given target. Those binding scaffolds may be used for a variety of applications, such as the construction of multispecific antibodies by attaching them to full-size IgG, intracellular targeting, affinity chromatography or detection assays. Desired properties of those scaffolds include high stability, high expression yield, small size, high solubility, low aggregation and negligible stickiness. Therefore, in the present study we aim to develop a novel binder scaffold based on a small, highly stable protein from a hyperthermophilic organism. In addition to being highly stable, extremely pH resistant and highly soluble, this protein also lacks cysteines. This property allows low-cost expression in E. coli, as well as usage for intracellular targeting applications. First, we will further improve the biophysical properties of this protein by computational design. This will be done in collaboration with Bruce Tidor, whose lab is located at MIT and who has already successfully collaborated with the Wittrup-lab (the host lab for this study, also located at MIT) in various previous studies. Next, the optimized protein will be expressed as a fusion to IgG in order to prove that this novel scaffold is suitable for constructing multispecific antibodies. Subsequently, based on the optimized scaffold, a library containing mutated surface residues will be constructed and displayed on yeast. By using a novel strategy for library design it will be possible to sample all possible binding domains in one yeast surface display library. This library will be used for selection of binders against a variety of model antigens. Finally, selected clones will be tested for usability in affinity chromatography, as well as for intracellular targeting. Importantly, our developed binders will not only target the intracellular antigen by simply binding to it, but we will develop a new system for targeted intracellular protein degradation. We hypothesize, that the library based on the optimized scaffold will enable the rapid development of highly stable binders. Those binders will be highly useful for the construction of multispecific antibodies, as well as for intracellular targeting applications. Moreover, due to the possibility of low-cost expression in E. coli, binders selected from this library will represent a low-cost alternative to antibodies in various biotechnological applications, such as detection assays (e.g. ELISA) and affinity chromatography.
Project staff
Paul Georg Furtmüller
ao.Univ.Prof. Dipl.-Ing.Dr.nat.techn. Paul Georg Furtmüller
paul.furtmueller@boku.ac.at
Tel: +43 1 47654-77277
Project Leader
01.01.2016 - 18.01.2016
Michael Traxlmayr
Priv.-Doz. Dipl.-Ing.Dr. Michael Traxlmayr
michael.traxlmayr@boku.ac.at
Tel: +43 1 47654-77274
Project Leader
19.01.2016 - 31.12.2016