Stable high-copy-number bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli.
The construction of new high-copy-number (hcn) lambda-promoter expression vectors is described. All these vectors (1) contain tandem lambda pR and pL promoters upstream of an extensive multiple cloning site (MCS) for insertion of genes, (2) direct expression of the lambda cIts857 gene, enabling their use in any Escherichia coli host strain for thermal induction of gene overexpression, and (3) bear the par locus of plasmid pSC101, ensuring their stable maintenance at hcn in the absence of continuous antibiotic selection. Six of the vectors also contain efficient ribosome-binding sites upstream of unique HpaI or NdeI sites in their MCS regions, and two contain sequences that encode N-terminal poly-His. The performance of these vectors was assessed by using them to overproduce the E. coli HMP flavohaemoprotein and the bacteriophage M13 gene II replicator protein.
Love
C A
CA
Centre for Molecular Structure and Function, Research School of Chemistry, Australian National University, Canberra, Australia.
Lilley
P E
PE
Dixon
N E
NE
D001426:Bacterial Proteins / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004279:DNA, Viral; D004926:Escherichia coli / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors; D006420:Hemeproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic
D001426:Bacterial Proteins; D004279:DNA, Viral; D006420:Hemeproteins; C086193:flavohemoprotein, Bacteria