Molecular Cloning of a Fluorescence‐Based Reporter Plasmid for Cell‐Free Protein Synthesis

Abstract

Cell‐free protein expression, also known as in vitro protein synthesis is a convenient and fast method for protein production. It bypasses the need to culture living cells, which can be time and resource consuming. Among multiple cell‐free protein expression systems available, the wheat germ extract system has become a popular choice for producing eukaryotic proteins due to high yields and commercial availability. The goal of this work was to generate a fluorescence‐based reporter plasmid to be used in wheat germ based cell‐free protein expression system. Because of its unique optical properties, the Green Fluorescent Protein (deGFP) can be used as a positive control during high‐throughput synthesis of proteins by a cell‐free system. To construct the reporter plasmid, Gibson Assembly strategy was used to clone a deGFP gene in a wheat germ extract compatible plasmid. The deGFP gene was successfully cloned in two plasmids, bearing a hexahistidine tag on either the N‐, or C‐terminus of GFP. The clones were validated by Sanger DNA sequencing. After validation, protein expression was confirmed by western blotting using an anti‐His antibody. The reporter plasmid will be used as a positive control during high‐throughput production of eukaryotic transcription factors by wheat germ cell free protein synthesis.Support or Funding InformationResearch Initiative for Scientific Enhancement (RISE), GRANT 5R25GM061151‐17This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.