Stable high-copy-number bacteriophage λ promoter vectors for overproduction of proteins in Escherichia coli

• Published in: Gene Vol. 176; no. 1; pp. 49 - 53
• Main Authors: Love, Christopher A., Lilley, Penelope E., Dixon, Nicholas E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 17-10-1996
• Subjects: Bacterial Proteins - genetics; Bacteriophage lambda - genetics; Base Sequence; DNA, Viral; Escherichia coli; Escherichia coli - metabolism; Expression vector; Gene cloning; Gene Dosage; Gene Expression; Gene II protein; Genetic Vectors; Hemeproteins - genetics; HMP protein; Molecular Sequence Data; Phage lambda; Plasmids; Promoter Regions, Genetic; A; RBS; SDS; SSB; nt; HMP protein; HMP; PAGE; Gene II protein; bp; Ap; p; hcn; LB; kb; Pollk; Gene cloning; NTA; ENase; Phage lambda; MCS; Expression vector; ts
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(96)00208-9

The construction of new high-copy-number (hcn) λ-promoter expression vectors is described. All these vectors (1) contain tandem λ
p
R and
p
L promoters upstream of an extensive multiple cloning site (MCS) for insertion of genes, (2) direct expression of the
λcI
ts857 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.