Generating temperature sensitive inteins for studying gene functions

Abstract

Reversible and easy to use, temperature-sensitive (TS) mutations, are powerful tools to study the functions of all genes, including essential and pleiotropic ones. However, the rarity of TS alleles and the difficulty of generating and identifying them have limited their use. A novel approach to generate TS mutations efficiently utilizes a conditionally-splicing intein, an "intein switch". Unmodified, an intein, which is a selfsplicing protein sequence embedded in-frame within a precursor protein, excises itself precisely from the precursor protein while simultaneously ligating the flanking sequences (exteins). The intein switch is a TS-intein that splices itself only at the permissive temperature (18°C) to generate a wild-type host protein. At the non-permissive temperature (30°C), it fails to splice and remains within the host protein, leading to loss of- function of the host protein. To extend the versatility of the TS-intein switch, we designed a scheme to look for intein switches that would function at different temperature ranges and that would splice with a high efficiency. We have generated and characterized 44 TS intein alleles. The permissive temperatures of these TS-intein switches differ from 18°C to 30°C. They function similarly in different host proteins. This makes it possible to choose a TS-intein switch according to the specific temperature determined by the optimal growth of an organism or a special experimental design.