[Construction and application of an Escherichia coli high effective expression vector with an enhancer]

In this study, we constructed a high effective fusion expression-vector in E. coli. This vector, pTO-T7, was characterized as: (1) an enhancer from tobacco mosaic virus (TMV), omega sequence, was ligated in front of a T7 promoter in the regulatory sequence; (2) the multi-cloning sites include eight restriction enzyme sites. It can facilitate fusion or nonfusion expression; (3) the N terminal of a fusion protein starts with the first 12 amino acids of T7 gene 10, and the C terminal is the hexahistidine tag; (4) kanmycin resistance gene was used as a selective marker. EGFP gene was inserted into pTO-T7 vector as a reporter gene. Expression data showed that fused-EGFP accounted to more than 50% of the total E. coli protein, and more than 90% of which was soluble. The fluorescence characters of fused-EGFP were also studied. The expression yield of target gene from plasmid pTO-T7 compared with that from pT-T7 without omega sequence suggested that omega sequence in pTO-T7 can improve the expression of target gene significantly.