Rapid purification of the oxygenase component of toluene dioxygenase from a polyol-responsive monoclonal antibody.
A monoclonal antibody designated 302 beta that is specific for the beta subunit of the oxygenase component (ISPTOL) of toluene dioxygenase from Pseudomonas putida F1 was used to prepare an immunoaffinity column. ISPTOL in cell extracts of Escherichia coli JM109(pDTG611) bound to the column, and an enzyme-linked immunosorbent elution-screening assay with different combinations of polyols and kosmotropic anions was used to determine the conditions necessary for recovery of active enzyme. Elution from an 8-ml antibody column with 50 mM 2-(N-morpholino)ethanesulfonate buffer (pH 6.8) containing 50% ethylene glycol, 1.0 M ammonium sulfate, 1.0 mM dithiothreitol, and 0.2 mM ferrous ammonium sulfate gave approximately 2 mg of ISPTOL with a specific activity that was more than 300 times the specific activity previously obtained.
Lynch
N A
NA
Department of Microbiology, College of Medicine University of Iowa, Iowa City, USA. nancy-lynch@uiowa.edu.
Jiang
H
H
Gibson
D T
DT
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D005260:Female; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D010105:Oxygenases / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D011108:Polymers; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology
D000911:Antibodies, Monoclonal; D011108:Polymers; C024617:polyol; D010105:Oxygenases; C023172:toluene dioxygenase