Optimizing Protein Degradation and Improving Energy Regeneration in Escherichia coli Cell-Free Systems and Developing A Simple Method to Dual Site-Specifically Label a Protein Using Tryptophan Auxotrophic Escherichia coli

This dissertation consists of two parts: the first is on optimization and improving protein synthesis and degradation in E. coli cell-free systems, and the second is about a method for incorporating 5-hydroxytryptophan into E. coli release factor 1 protein (RF1) for dual site-specific labeling. Protein synthesis and degradation are fundamental processes in all living cells–the former creates all the protein required for biochemical and cellular functions, and the latter is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. Here we are interested in creating synthetic genetic circuits that function in a cell-free expression system, which will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We further showed that adenosine monophosphate (AMP) accumulates in the cell-free systems, reducing the availability of adenosine nucleotides to energy regeneration. We use class III polyphosphate kinase 2 from Meiothermus ruber (MrPPK2) to mitigate AMP accumulation and improve the performance of protein synthesis and degradation in the cell-free systems. Site-specifically labeling proteins with multiple dyes or molecular moieties is an important yet not-trivial task for many researches, such as when using Föster resonance energy transfer (FRET) to study dynamics of protein conformational change. Many strategies have been devised, but usually done on a case-by-case scenario. Expanded genetic code provided a general platform to incorporate non-canonical amino acids (ncAA), which can also enable multiple site-specific labeling, but it’s technically more complicated and not suitable for some specific researches which are in conflict with its technical foundation. We want to develop an easy-to-use protocol for site—specific dual labeling by incorporating 5-hydroxytryptophan using tryptophan auxotroph strain of E. coli, which can provide an extra orthogonal bioconjugation handle additional to conventional amine- or thiol-based labeling method. As demonstration, we incorporated 5-hydroxytryptophan into E. coli release factor 1 (RF1), a protein known to possess two different conformations, and labeled two different fluorophores specifically on 5-hydroxytryptophan and cysteine site. This method provides an easier way to achieve dual- or multi-labeling of protein that will be useful for biochemical or biophysical experiments like FRET, which can help us study the detailed mechanism of RF1-mediated translation termination