Plasmid vector for overproduction and export of recombinant protein in Escherichia coli: efficient one-step purification of a recombinant antigen from Echinococcus multilocularis (Cestoda).
We describe the use of the Escherichia coli plasmid vector, pVB2, for high-level expression and export of recombinant protein. The pBR322 derivative pVB2 harbors the mglB gene, which codes for the galactose-binding protein (GBP) of E. coli. GBP is exported into the periplasmic space of the bacterial cell. Gene mglB contains an EcoRI restriction site close to its 3' end which allows simple in-frame insertion of EcoRI fragments obtained from recombinant lambda gt11 phages. The pVB2 vector was used to express an antigen from Echinococcus multilocularis. The recombinant protein amounted to over 50% of total cellular protein and could be efficiently isolated from the periplasm by osmotic shock. The application of the purified antigen in an ELISA enabled a clear and specific detection of anti-Ec. multilocularis antibodies in human patients' sera, which had been immunosorbed with a periplasmic extract (containing wt GBP) before investigation. These data show the general usefulness of pVB2 as an expression vector for producing in E. coli diagnostically relevant antigens from any infective organism.
Müller
N
N
Institute of General Microbiology, University of Berne, Switzerland.
Vogel
M
M
Gottstein
B
B
Scholle
A
A
Seebeck
T
T
D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D010582:Bacteriophage lambda / Q000235:genetics; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004446:Echinococcus / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*
D000947:Antigens, Helminth; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C023550:galactose-binding protein; D005947:Glucose