Low-copy-number T7 vectors for selective gene expression and efficient protein overproduction in Escherichia coli.
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 beta-galactosidase (beta Gal) and UTP:alpha-D-glucose-1-phosphate uridylyltransferase (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.
Dersch
P
P
Max-Planck-Institut für Terrestrische Mikrobiologie, Marburg, FRG.
Fsihi
H
H
Bremer
E
E
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011991:Receptors, Virus; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011991:Receptors, Virus; C066743:Tsx protein, E coli; D001616:beta-Galactosidase