Isolation of recombinant mycobacterial antigens by an automatic and generally applicable purification method for beta-galactosidase fusion proteins.
An automated two-dimensional chromatographic method has been developed for the isolation and concentration of recombinant fusion proteins with beta-galactosidase. The system consists of an immunoaffinity column with anti-beta-galactosidase antibodies as ligand, followed by an anion-exchange column. It was used for the purification and concentration of recombinant fusion proteins from Mycobacterium tuberculosis and M. leprae. Small amounts of crude lysates of Escherichia coli were loaded stepwise onto the immunoaffinity column with intermittent washing, elution and re-equilibration. After several cycles the eluate was passed through the anion-exchanger. Using an immunoaffinity gel of 5-ml volume and the anion-exchanger Mono Q HR 5/5, from 10 ml of crude E. coli lysate (containing up to 50 mg of protein) up to 100 micrograms of recombinant protein in a 2-ml volume could be isolated overnight.
Schoel
B
B
Max-Planck-Institut für Immunbiologie, Freiburg, F.R.G.
Kaufmann
S H
SH
D000907:Antibodies, Bacterial / Q000302:isolation & purification; D000942:Antigens, Bacterial / Q000302:isolation & purification*; D001322:Autoanalysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000302:isolation & purification*; D009161:Mycobacterium / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000302:isolation & purification*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005696:Galactosidases; D001616:beta-Galactosidase