Specific immuno-detection of benzoate-para-hydroxylase with antibodies raised against synthetic peptides.
As a model system for the industrial use of fungal cells in the enzymatic conversion of chemicals, the parahydroxylation of benzoate was studied. To increase the amount of benzoate-para-hydroxylase (BPH, EC 1.14.13.12.) in the cell the gene coding for the enzyme (bphA) was cloned and expressed in Aspergillus niger. Detection of the enzymatic activity of the protein was not reproducible. It was decided to raise an antiserum for immuno-detection purposes. Sufficient benzoate-para-hydroxylase for immunization could not be obtained; therefore the synthetic-peptide strategy was used. We demonstrate that synthesis of antigenic determinants, can be useful in the production of highly specific reagents for the detection of proteins. The availability of monospecific polyclonal sera opens new possibilities in functional studies and purification of benzoate-para-hydroxylase.
Medical Biological Laboratory TNO, Rijswijk, The Netherlands.
K
K
Gerritse
R
R
Van Gorcom
M
M
Fasbender
J
J
Lange
M
M
Schellekens
N
N
Zegers
E
E
Claassen
W
W
Boersma
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D050565:Benzoate 4-Monooxygenase; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005260:Female; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology*; D011817:Rabbits
D000906:Antibodies; D000939:Epitopes; D010455:Peptides; D006899:Mixed Function Oxygenases; D050565:Benzoate 4-Monooxygenase