Open AccessLow‐copy‐number T7 vectors for selective gene expression and efficient protein overproduction in Escherichia coli
Author(s) -
Dersch Petra,
Fsihi Hafida,
Bremer Erhard
Publication year - 1994
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1994.tb07195.x
Subject(s) - t7 rna polymerase , biology , ribosomal binding site , gene , microbiology and biotechnology , plasmid , expression vector , cloning vector , transcription (linguistics) , genetics , multiple cloning site , start codon , escherichia coli , gene expression , molecular cloning , bacteriophage , ribosome , rna , messenger rna , recombinant dna , linguistics , philosophy
A set of low‐copy‐number vectors (pPD) has been constructed that permit selective gene expression and high‐level protein overproduction in Escherichia coli , based on the bacteriophage T7 RNA polymerase/T7 promoter system. These plasmids carry a chloramphenicol resistance gene ( cat ) as a selective marker and an extended multiple cloning site for convenient gene cloning. Their replication is mediated by ori sequences derived from the low‐copy‐number vector pSC101. The efficient T7 gene 10 promoter present on these vectors allows selective and high‐level transcription of cloned genes carrying their own translational initiation signals. In addition, low‐copy‐number T7 vectors were constructed that permit expression of genes lacking their own transcription and translation initiation elements by providing a ribosome binding site, an ATG start codon and a multiple cloning site devised for the cloning in all three reading frames. The pPD expression vectors were used to achieve high‐level overproduction of the E. coli integral outer membrane protein Tsx, and the cytoplasmic enzymes β‐galactosidase (βGal) and UTP:α‐d‐glucose‐1‐phosphate uridylytransferase (GalU). The characteristics of these low‐copy‐number T7 expression vectors should prove very useful for the cloning and high‐level overexpression of genes whose gene products are deleterious to the E. coli host.