Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12.
The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.
Wood
J M
JM
Department of Chemistry and Biochemistry, University of Guelph, Ont., Canada.
Taylor
K A
KA
McClellan
D J
DJ
Lawrie
G G
GG
Krogsrud
R L
RL
Beveridge
T J
TJ
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000936:Antigen-Antibody Complex; D002462:Cell Membrane / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D006825:Hybridomas / Q000276:immunology; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000276:immunology*; D011394:Proline Oxidase / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011394:Proline Oxidase