Preliminary study on preparation of E. coli cell-free system for protein expression

Abstract

In the new era of “Omics”, the traditional techniques of protein expression in vivo can not come up with the exponential increase of genetic information. The cellfree protein synthesis system provides a new strategy of protein expression with advantages of rapid, convenient and high-throughput expression. The preparation of cell extracts, the optimization of substrate concentrations and the energy regeneration system are the key factors for the successful construction of cell-free protein expression system. In this work, the cell extract was prepared from RNase I- defective strain E. coli A19. The cell growth phase, the pressure for cell disruption and the storage condition of cell extracts were optimized. Meanwhile, the optimal substrate concentrations and the energy regeneration system were selected. Under the optimized conditions, the green fluorescent protein (GFP) reporter gene was expressed in the E. coli cell-free system with high expression level (Ca. 154 μg/mL) which was 29 times higher than the expression level before optimization.