Discovery, Design, and Deployment of Enhanced Split Inteins

Abstract

Protein splicing is a post-translational autoprocessing event in which an

intervening protein (intein) spontaneously cleaves itself from a precursor protein

while simultaneously ligating the adjacent residues (exteins) to form a native

peptide bond. Due to the efficiency of this ligation reaction, inteins have found

widespread use as tools in protein engineering and chemical biology. Naturally split

inteins, which are separately expressed and then undergo association and splicing in

trans, are of particular interest. However, applications of split inteins have been

limited by a number of shortcomings, including issues of expression yield among

protein-intein fusions and decreased splicing rates under certain extein contexts

(extein dependency). Through structural and mechanistic-guided approaches, we

have engineered split inteins with improved robustness and extein promiscuity. We

utilized consensus design to engineer a consensus fast (Cfa) split DnaE intein that

exhibits unprecedented thermal and chaotropic stability and a consensus atypical

(Cat) split intein that is the fastest atypical split intein reported to date. In addition,

targeted saturation mutagenesis was applied to engineer extein promiscuity into

DnaE inteins. Lastly, to address the tendency of split intein fusion proteins to

aggregate, a general strategy using intrasteric stabilization was employed in which

an appended peptide sequence acts as an internal chaperone for split intein and

extein fragments. These enhanced split inteins were then applied to a number of

protein engineering methods, such as the generation of head to tail cyclized

proteins, the modification of a monoclonal antibody with a small molecule cargo,

and the semisynthesis of cellular chromatin in isolated nuclei. Furthermore, the

increased stability of Cat compared to previously reported atypical split inteins

enabled the first mechanistic investigation of atypical split intein association.

Overall, we expect these enhanced inteins to improve current intein-based

technologies and encourage the further use and development of protein trans-

splicing as a tool for protein engineering.