Industrial protein production with highly efficient ncAA incorporation
Patent #22005/TUB
Background
The expansion of the genetic code using orthogonal aminoacyl-tRNA synthetase/tRNA pairs is an established tool in synthetic biology. However, known PylRS-based systems suffer from low catalytic efficiency, especially in the multisite incorporation of ncAAs. This limits both yield and industrial applicability.
Technical Description
The present invention addresses this limitation by developing a highly active, psychrophilic PylRS variant from M. burtonii with targeted mutations in the catalytic centre. This makes it possible to specifically equip recombinant proteins with additional chemical functions that are not accessible or only very inefficiently accessible using conventional biotechnological methods. During protein production in the cell, the greatly improved enzyme ensures that so-called non-canonical amino acids (ncAAs) are reliably and efficiently incorporated into proteins. These ncAAs carry special chemical groups, e.g. for coupling reactions, labelling or light-active functions.
The efficiency of the technology has already been validated in E. coli and in human HEK293T cells.
Possible Applications
• Production of recombinant proteins with defined chemical functions, e.g. photoreactive, click chemical or conjugatable groups
• Protein-drug conjugates (PDCs) and protein-protein conjugates
• Site-specific labelling of proteins for biophysical and cell biological analyses
• Photo-crosslinking studies to investigate protein-protein interactions (e.g. using photo-leucine)
• Platform technology for synthetic biology, protein engineering and functional proteomics
