The aim of the project is that to develop an in vitro system which permits the efficient synthesis of proteins in vitro at high temperature. This method is based on the use of an unfractionated cell lysate (S-30) from Sulfolobus solfataricus previously well characterized in our laboratory and the novelty of use it coupled with an in vitro transcription system.
The novelty of this project is based on the use of an essential element corresponding to a strong promoter derived from 16S/23S rRNA-encoding DNA promoter from the archaebacterium Sulfolobus sp. P2 that produces, with high efficiency, specific mRNAs.
The simplicity of the experimental procedure and specific activity of the proteins produced offer a number of possibilities for the study of structure-function relationships of proteins.
Cell-free protein synthesis (CFPS) has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications.
In our laboratory, we have developed since a long time a CFPS from the thermophilic archaeon S. solfataricus. However, our standard system uses only pre-transcribed RNA templates, while CFPS from hyperthermophiles allowing coupled transcription-translation have not so far, to the best of our knowledge, been described. The aim of the project is that to set up a system of a coupled in vitro transcription/translation system for cell-free protein synthesis from the thermophilic archaeon Sulfolobus solfataricus. In other words, our intent is that to find the best conditions permitting to program Sulfolobus solfataricus whole cell lysate with a plasmid containing a gene of interest favoring its transcription and coupled translation to the same time.
The most obvious result will be that to tune a powerful tool to expand our understanding of the molecular mechanisms governing coupled transcription-translation in archaea. Moreover, the production of thermostable proteins for biochemical and crystallographic characterization will be favored by natural conditions of high temperature permitting the correct fold into their native state.
The advantage of this CFPS systems over in vivo methods will be that one can dispense with all the procedures required to support cell viability and growth. At the same time, the simplicity and low cost of preparing cellular extracts make the system a preferential choice among the available tools for the synthesis of proteins of interest.
The assessment of the reaction conditions will be carried out in small volumes for obvious reasons and they will not allow the production of proteins in large amount. However, we are confident that the setting of different parameters and components that affect the rate and yield of protein synthesis will be useful as a reference and start point in the future for the developing of a technological platform for industrial and high-throughput protein production. Indeed, the main advantages derived from the expression of the proteins at high temperatures are reduced risk of contamination, lower viscosity, higher solubility of substrates and improved transfer rates. This means that the high demands of the biotech industries and the rapid development of new techniques such as metabolomics and direct evolution will make our system a viable choice for new industrial processes.