The Construction Of Cell-Free System Derived From E. Coli Or Silk Gland

With the rapid development of bio-technology, substantial sequenced genes demand a time-saving and high-throughput method to synthesize proteins. Cell-Free protein synthesis system has attracted extensive attention due to its high ability of protein expression, simplicity of operation and the potential for high-throughput protein synthesis. The aim of this work is to establish an economical method to produce E. coli cell-free extract with high translatian capacity by improving the preparation procedure. Also, the silk gland of Bombyx mori was utilized to construct a novel cell-free protein synthesis system, which expands the the source of organisms to pruduce cell-free extract. The silk gland cell-free system is promising to investigate eukaryotic proteins.In this work, the eGFP, as a report gene, was cloned into the plasmid of pIVEX2.4c to construct the plasmid of pIVEX2.4c-eGFP, which was used as the expression template in E. coli cell-free protein synthesis system. E. coli BL21(DE3) was selected to produce the cell-free extract. Its growth curve was measured and logarithmic growth phase was determined.The procedure to produce E. coli extract was optimized to reduce the labor work and cost, and the high ability of protein expression was kept. The amount of eGFP synthesized in this E. coli cell-free system could reach to 427μg/mL. This is the first report in China on establishing an efficient E. coli cell-free system by improving the extract preparation method. Besides, a novel cell-free protein synthesis system was constructed from silk gland of Bombyx mori. In this system, Luciferase ICE T7 Control Vector was the expression template, the concentration of main components was optimized, and the amount of Luciferase synthesized could come up to 1.2μg/mL, which was equivalent to commercial Rabbit Reticulocyte Lysate. At last. the native promoters of silk gland were studied for protein synthesis in this silk gland cell-free system.