Construction and application of a novel plasmid "ATG vector" for direct expression of foreign genes in Escherichia...

A new type of plasmid expression vector was developed for direct expression of foreign genes in Escherichia coli. The plasmid vector, designated pTrS3, carries the E. coli tryptophan (trp) promoter and the Shine-Dalgarno (SD) sequence for the trp leader peptide as well as an ATG sequence located 13 bp downstream from the SD sequence. The dG residue of this ATG overlaps with the first dG residue of the single Sph I recognition sequence (GCATGC) of the vector DNA. After cleaving pTrS3 DNA by Sph I, the 3' protruding Sph I ends were converted into blunt ends using the Klenow fragment of E. coli DNA polymerase I. Subsequently, the DNA fragments coding for mature human interferon-beta or for the interferon lacking several aminoterminal amino acids, were ligated to this vector DNA and cloned in E. coli. Interferon activity was detected in the extracts of bacterial strains harboring the recombinant plasmids and the results indicated that the interferon-beta polypeptides without the five aminoterminal amino acids might be less active than the mature form.