Mémoire
| Résumé : | The stringent response is a mechanism to palliate different environmental stresses in bacteria. This mechanism is regulated by secondary messengers (p)ppGpp levels which are controlled by the RSH superfamily of enzymes. These enzymes are responsible for synthetising and hydrolizing (p)ppGp. Within this family of enzymes, there are the long RSH with notably Rel and RelA. These proteins (~80 kDa) are too small and have a preferential orientation issue to achieve high resolution 3D maps of the enzyme by Cryo-EM. In order to achieve that, Dr. Abel Pino-Garcia has developed a construct called the Spear. It is a synthetic protein made of a nanobody, a 3 helix bundle (PDB identifier: 4TQL) and a His-tag.This project aims to develop a protocol to express and purify a synthetic protein – the Spear – that would bind to the targeted RSH enzyme and facilitate its structural analysis by Cryo-EM.Our results show that the first construct with Nanobody96 targeting Escherichia coli RelA -Spear96- was not viable in E. coli. Another construct with Nanobody585 -Spear585- was viable in E. coli. For this construct a ‘heat shock’ expression and purification protocol was established. The Spear585 was then purified in complex with RelCtep but the sample was not pure enough to analyse it by Cryo-EM. In order to see how the Spear585 influences the Cryo-EM analyses it would be a priority to purify the complex to do a Cryo-EM analyses. |
