Development of high-performing, cysteine-less split inteins as versatile tools for new protein engineering applications

• Applicant: Professor Dr. Henning D. Mootz
• Subject Area: Biochemistry ; Biological and Biomimetic Chemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545035351

Inteins remove themselves out of a precursor protein by catalyzing the multi-step protein splicing reaction, in which the N- and C-terminal flanking sequences (exteins) are linked with a native peptide bond. Protein splicing is virtually traceless and strictly requires only a single residue (cysteine, serine or threonine) to remain at the ligation site. In split inteins the intein domain is separated into two fragments, which perform protein trans-splicing between two precursor proteins following association and folding. Split inteins in particular have enabled a variety of unique techniques to manipulate the structure and function of proteins on the posttranslational level. Key advantages are the inherent affinity and the traceless nature of the reaction. This proposal deals with cysteine-less split inteins that constitute a very minor fraction of all known inteins. Cysteine-less inteins are devoid of cysteine residues and do not depend on cysteine side chains for their acyl rearrangement reactions during protein splicing. Cysteine-less split inteins have only very recently come into the focus as especially useful tools for protein engineering. They can perform their protein trans-splicing reaction in the absence of any reducing agents and in oxidizing environments. This key property is of immense practical importance, for example when dealing with purified proteins that contain sensitive disulfide bonds, or when aiming to engineer proteins on the surface of mammalian cells without causing collateral damage to countless disulfide-stabilized receptor proteins. Previously, we have reported the first two cysteine-less split inteins that are in principle suitable for general protein engineering. However, their usefulness has been limited due to impaired folding and splicing properties, respectively. In this proposal, we plan to improve both cysteine-less split inteins by rational protein design and directed protein evolution to obtain highly optimized variants. These high-performing protein ligation tools will be widely applicable to protein engineering. Furthermore, we will demonstrate their potential by developing a set of novel and innovative protein engineering methods. These will add powerful new opportunities, for example for minimally invasive labeling of cell-surface proteins, multi-site selective protein labeling, multi-site incorporation of different unnatural amino acids, and the first intein-mediated protein-protein conjugation via an amino acid side chain. Finally, just as our strategies to improve the split intein tools also rely on the first insights into the specialized mechanism of cysteine-less protein splicing, we will further our understanding of the differences in sequence and mechanism to their cysteine-dependent counterparts.

• DFG Programme: Research Grants