A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase act...

Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on ad...

A method to photo-chemically trigger fluorescent labelling of proteins in live cells is developed. The approach is based on photo-caged split-intein mediated conditional protein trans-splicing reaction and enabled background-free fluorescent labelling of target proteins with the necessary spatiotemporal control.

Fluorescence resonance energy transfer (FRET) is an ideal tool for studying protein conformational changes and dynamics. Strategies to specifically label proteins with multiple colors at structurally important sites, a prerequisite for such studies, however, need to be devised on a protein-by-protein basis. Here we describe a simple yet widely applicable two-color site-specific protein labeling...

The genome of the obligate intracellular pathogen Coxiella burnetii contains a large number of mobile genetic elements including two group I introns and an intervening sequence (IVS) that interrupt the 23S rRNA gene; an intein within dnaB (encoding replicative DNA helicase) and a homing endonuclease. The introns are self-splicing ribozymes and able to inhibit ribosome function and retard bacter...

The hyperthermophilic archaeon Thermococcus litoralis strain NS-C, first isolated in 1985, has been a foundational organism for archaeal research in biocatalysis, DNA replication, metabolism, and the discovery of inteins. Here, we present the genome sequence of T. litoralis with a focus on the replication machinery and inteins.

A genetically encodable caspase-3 sensing system has been created using self-assembling split-GFP, in which a C-terminal fragment is "covalently" cyclized via a caspase-3 substrate sequence mediated by split-intein. The specific cleavage of the cyclic C-terminal fragment by caspase-3 induces the GFP reassembly and fluorescence recovery.

Project aims 1. Feasibility studies of Bacillus subtilis as an expression host for the production of therapeutically important proteins through high cell density fermentations. 2. Studies on intein mediated purification of therapeutically important proteins expressed in Bacillus subtilis. 3. Case studies with a few proteins of research/commercial relevance including malaria proteins that are li...

............................................................................................................................... i Acknowledgements ............................................................................................................. ii Table of