THESIS
2018
xii, 147 pages : illustrations (some color) ; 30 cm
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 en...[
Read more ]
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.
Post a Comment