Research Projects

Abstract

The commercial use of proteins, including enzymes and biotherapeutics, is often limited by their low intrinsic stability and inherent loss of catalytic activity. Existing approaches to protein thermostabilisation can be broadly divided into i) directed evolution of proteins and ii) rational protein design. Directed evolution is based on the principles of natural evolution, generating a large number of randomly mutated variants of the gene encoding the target protein, expressing them and subjecting them to appropriate selection methods to identify thermostabilised protein variants. Rational protein design, in contrast, relies on the targeted substitution of predefined amino acid residues to increase the number of stabilizing interactions within the protein. Despite advances in computational algorithms for protein design, directed evolution remains a powerful tool for protein thermostabilisation. The potential of this approach has not yet been fully exploited, as the capacity of existing methods to select thermostable proteins from vast libraries of random protein mutants is limited.

In this project, we will improve the throughput of directed protein evolution towards increased thermostability by coupling mRNA-display of massive number of randomly mutated protein variants with selection of protein conformational stability at elevated temperatures. To achieve this, we will modify the intracellular proteases of thermophilic microbes, whose biological function is the degradation of incorrectly folded or damaged proteins. We propose that these proteases will degrade less stable (conformationally disordered) variants of the target protein at elevated temperatures, while leaving stable variants intact. Non-degraded, thermostable protein variants will then be captured onto solid particles via specific tags and the corresponding mRNAs will be reverse transcribed and amplified. Through several cycles of mRNA-display and proteolytic selection, the DNA library will be enriched with thermostable variants. The model protein used for development of this method will be PETase, an enzyme that degrades polyethylene terephthalate. This enzyme has high applicative value, as its substrate (PET plastics) is one of the most problematic environmental pollutants. To obtain thermostabilised PETases with retained or improved catalytic activity, we will subject the PETase DNA library enriched with thermostabilized variants by the developed mRNA-display approach to further activity-based screening. For this, we will integrate multiplex cell-free protein synthesis and subsequent in vivo single colony screening. The thermostabilised PETases will be biochemically and biophysically characterised in terms of their thermal stabilities and activities towards PET substrates to further elucidate the effects of stabilizing mutations. The novel approaches developed in the scope of this project will expand our understanding of the thermal adaptation mechanisms of PET-degrading enzymes and provide advanced strategies for developing industrially relevant enzymes and other proteins with desired thermostabilities.

External link to Researchers Open in new window

University of Ljubljana, Biotechnical faculty:

• Department of Food Science and Technology, Chair of Biochemistry and Food Chemistry 
• Department of Biology, Biochemistry Research Group 
• Department of Wood Science and Technology, Wood Pathology and Preservation 

University of Maribor, Faculty of Chemistry and Chemical Engineering:

• Laboratory of Physical Chemistry and Chemical Termodynamics

The phases of the project and their realization

Phase 1 (month 1 – month 8): Identification and modification of proteases for selecting thermostable from unstable proteins at elevated temperatures

Phase 2 (month 8 – month 28): Implementation of mRNA-display, coupled with thermostabilized PETase selection with proteases

Phase 3 (month 28 – month 36): Characterisation of thermostabilized PETases

Project partners

• UM FKKT