Construction and optimization of cell-free protein expression platform for in vitro synthesis of phosphoprotein

Abstract

Cell-free protein synthesis (CF-PS) system is widely used technique for the production of recombinant protein without using intact cells. It utilizes biomolecular machineries extracted from cell and enables the expression of desired protein from template DNA with the gene of interest in cell-free environments. Traditional systems for recombinant protein expression mostly depend on in vivo conditions using bacteria, yeast and mammalian cells. The limitations of these systems include demanding maintenance for cell viability conditions and higher production cost. CF-PS system, on the other hand, is advantageous for efficient protein expression in that researchers can reconstitute the components and manipulate in vitro conditions without concerning cell viability. Therefore, it facilitates the optimization of protein expression conditions and focuses the use of most energy sources in cell-free condition on target protein synthesis. In this respect, CF-PS has emerged as a potent tool for the advance of protein synthesis technology. We first constructed CF-PS platform that are applicable for a wide range of protein synthesis with consistency using two kinds of Escherichia coli strains, BL21(DE3) and C321 ∆A expanded. Then, we tested the system by validating the synthesis of super-folder green fluorescen protein (sfGFP). After the establishment of stable platform, we next attempted the changes in the step of crude cell extract preparation to assess cell extract performance in CF-PS reaction. Then, our works continued to the manipulation of cell-free conditions to search for optimized protein expression environments. These optimization processes belong to the use of different secondary metabolites as phosphate source and introduction of nucleoside triphosphate (NTP) regeneration system composed of a variety of kinases. In the final step of our research, our CF-PS system applied to site-specific incorporation of non-standard amino acid, phosphoserine into synthesized protein by amber codon replacement. Here, we established and validated our platform that has the potential for in vitro synthesis of proteins with diverse functions.