Cell-free synthesis and purification of recombinant nucleocapsid (N), membrane (M), and envelope (E) proteins

Rapid production of soluble recombinant antigens is important for developing in vitro diagnostics, vaccines, and drugs against virus such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this research, hard-to-express nucleocapsid, membrane, and envelope proteins were successfully expressed by an Escherichia coli-based cell-free protein synthesis system. The solubility of the proteins was optimized using various amphipathic molecules. Most of the impurities were easily removed by a one-step Ni-NTA affinity chromatography. This study provides an easy and quick alternative for virus’s trans-membrane and nucleotides associated recombinant protein expression, which has potential downstream application for early screening of newly emerging viruses.