Construction and characterization of a highly regulable expression vector,pLAC11, and its multipurpose derivatives, pLAC22 and pLAC33

A number of different expression vectors have been developed to facilitate the regulated overproduction of proteins in Escherichia coli and related ba cteria. Some of the more popular ones include pKK223-3, pKK233-2, pTrc99A, and the pET family of expression vectors. These vectors were designed to be regulable and can be grown under conditions, that repress protein producti on or under conditions that induce protein production. Unfortunately, howev er, numerous researchers have found that these vectors produce significant amounts of protein even when grown under repressed conditions. We describe here a new expression vector, pLAC11, which was designed to be more regulab le and thus more tightly repressible when grown under repressed conditions. The tight regulation of pLAC11 was achieved by utilizing the O3 auxiliary operator. CAP binding site, promoter, and O1 operator that occur in the wil d-type lac control region. The pLAC11 vector can be used to conduct physiol ogically relevant studies in which the cloned gene is expressed at levels c omparable to that obtainable from the chromosomal copy of the gene in quest ion. In experiments in which a bacterial cell contained both a null allele in the chromosome and a second copy of the wild-type allele on pLAC11, we o bserved that cells grown under repressed conditions exhibited the null phen otype. Two multipurpose derivatives of pLAC11, pLAC22, and pLAC33 have also been constructed to fulfill different experimental needs. (C) 2000 Academi c Press.