| Inteins are internal elements that can be found in organisms in all three domains of life and offer a number of attractive methods for biotechnology applications. The applications of intein include tagless protein purification, biosensors, and tools for pharmaceutical research. However, there are some drawbacks limiting the applications of intein. Intein's trans-splicing reactions can be a powerful tool on the protein engineering field, but split inteins usually have low splicing efficiency. Also, the reassembly of exteins usually involves only two domains; the assembly of more than two exteins cannot be accomplished. A focus in our lab, therefore, has been to overcome its limitations to make intein a more versatile tool and to discover new applications for intein. We first developed a reporter assay based on the subcellular localization of the reverse tetracycline-controlled transcriptional activator (rtTA) to engineer Nostoc punctiforme PCC73102 DnaE split intein into a temperature sensitive intein as well as to improve its splicing efficiency. Second, we developed a novel intein system that can be applied to splice multiple-domain proteins. Trans-splicing is used to implement novel split intein mediated modular synthesis (SIMMS) to allow the post translational splicing of more than two exteins. These newly developed strategies significantly enhance the utility of inteins in biotechnology studies. |
