Application of inteins to the production of commercially valuable proteins and studying of intein-like proteins

Abstract

Inteins, genetic elements which are similar to self-splicing introns, have been widely studied and utilized for academic research as well as biotechnological applications. The unique feature of inteins as a self-splicing/cleavage element has enabled the development of methods for tag-free recombinant protein production, protein labelling, and peptide ligation. Despite the great potential of inteins in protein engineering, the mechanism for intein splicing/cleavage is still not fully understood. Lacking of a comprehensive understanding of this intein mechanism is a significant reason for the poor control of intein-mediated protein splicing/cleavage processes and is a factor that largely contributes to the limited use of inteins. To expand the applications of inteins in protein engineering, other than the widely used protein expression host E. coli, we also used Bacillus subtilis which is an intein-free bacterium for intein-fused protein expression, and it turned out that inteins can be fully functional in B. subtilis. Moreover, through mutagenesis studies it was discovered that C-terminal fragment of Ssp DnaB intein containing motif F and G are crucial to intein C-terminal cleavage activity and N-terminal fragment of Ssp DnaB can be dispensable for C-terminal cleavage. Our findings may not be only used for setting up a one-step tag-free protein purification, but it may also shed light on intein structure-function relationships. Intein-mediated cleavage is a process uncoupled from splicing which led us to wonder about the existence of protein domains with capability of self-cleavage but not splicing. It was later discovered that cellulose binding domain of cellulases was separated from the catalytic domain when expressed in E. coli. Its self-cleavage feature was confirmed when bFGF was fused to its C-terminus and can be precisely cleaved off from cellulose binding domain.

Date of Award	2018
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology