Protein engineering

Ultimately, we study the biophysical and structural features of proteins to achieve information for structure-based protein engineering, which we see as an attractive choice for creating new biotechnological and biomedical applications. 

Our research in protein engineering is focused on protein splicing and creating more practical, robust and adaptable splicing elements for engineering purposes, such as ligation techniques. Split Inteins are very useful tools in protein ligation and engineering, due to their ability to ligate two disconnected precursors into one functional sequence. 

Two essential qualities of split Inteins for engineering purposes are robust protein activity and high sequence tolerance at the splice junction. However, split Inteins that possess both of these attributes are fairly rare and in high demand. Especially orthogonal (Oth) split Inteins are desirable for one-pot multiple fragment protein ligation, or the PTS-based biorthogonal conjugation requiring several split inteins. 

Other very important factor in choosing an intein for engineering purposes, is the size of the intein. Generally, the smaller the intein, the less interference it causes, and therefore is preferable to larger intein sequences. 

One of the most frequently used naturally split Inteins is DnaE from Nostoc punctiforme, which tolerates various amino acid types at the splice junction. Research we’ve conducted has indicated the NpuDnaE to have various advantages to other types of split Inteins.

The main challenge of using more prevalent cis-splicing Inteins for engineering purposes in vitro is their poor solubility. In addition to naturally split inteins, we’ve studied methods to artificially create novel natural-like split Inteins from the more prevalent cis-splicing Inteins. One way of achieving this could be converting the cis-splicing Inteins by grafting a a charge network which is similar to naturally split Inteins. We’ve done research on this subject, and at least in one case, the regenerating a naturally split intein-like charge network was sufficient to yield a orthogonal split intein, though this approach has to be studied more.

Potentially, these methods could produce new orthogonal split Inteins with, for example high tolerance of foreign extein sequences or optimal junction sequences, which could be utilized in applying PTS techniques to a wider range of protein engineering applications.