An Escherichia coli plasmid vector system for high-level production and purification of heterologous peptides fused to active chloramphenicol acetyltransferase.
A very small plasmid vector system is described for construction and high-level production of C-terminal chloramphenicol acetyltransferase (CAT) fusion proteins in Escherichia coli. The only functional elements of the plasmid are a minimal region of the ColE1 origin of DNA replication and the Tn9 cat gene, both under control of a tac promoter. Since C-terminal fusion to CAT does not interfere with chloramphenicol (Cm) resistance, plasmids are maintained under Cm selection. Because of its small size (1392 bp), the system is especially convenient for building and expression of synthetic genes and gene fragments. This concept was utilized to generate a fusion with a synthetic gene encoding the multiple-epitope fragment from the rubella virus E1 membrane protein. Affinity-purified fusion proteins were obtained in mg amounts from 100-ml batches of culture fluid, and incorporated as a specific antigen in a rubella immunoglobulin G enzyme-linked immunosorbent assay.
Robben
J
J
Laboratory of Gene Technology, K.U. Leuven, Belgium.
Massie
G
G
Bosmans
E
E
Wellens
B
B
Volckaert
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012412:Rubella virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics
D004269:DNA, Bacterial; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C061826:E1 envelope protein, Rubella virus; D007544:Isopropyl Thiogalactoside; D015500:Chloramphenicol O-Acetyltransferase