Use of a lac promoter-operator fragment as a transcriptional control switch for expression of the constitutive lpp gene in Escherichia coli.
We constructed hybrid plasmids to allow controlled expression of the lpp gene coding for the outer membrane lipoprotein of Escherichia coli, which is otherwise expressed constitutively. This was achieved by the insertion of a DNA fragment carrying the lacUV5 promoter-operator region as a transcriptional control switch into the 5'-untranslated region of the lpp gene. When fully induced, the production of the lipoprotein, controlled under the tandem promoters of lppp-lacpo-lpp, increased approximately 3-fold compared to that under lacpo-lpp control. However, it was still only one-third of the lipoprotein production under the constitutive lpp expression. One such plasmid, pKEN125, carrying lppp-lacpo-lpp in pBR322 produced only a trace amount of the lipoprotein without induction in an E. coli lpp- cell. Upon the addition of isopropyl-beta-d-thiogalactoside, however, the amount of the lipoprotein reached almost 40% of the total membrane proteins. Cells carrying pKEN125 grew normally in the presence of the inducer, whereas cells carrying plasmid pKEN126 with tandem duplication of lppp-lacpo-lpp sequences in pBR322 lysed upon induction at high temperature. In cells with pKEN126 induced at high temperature, at least three new bands which were cross-reactive with antilipoprotein serum in addition to the mature lipoprotein were detected by pulse-labeling cells with [35S]methionine.
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D007763:Lac Operon; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D008565:Membrane Proteins; D004262:DNA Restriction Enzymes