Recombinant antibody fragments that detect enoyl acyl carrier protein reductase in Brassica napus.
Purified Brassica napus enoyl acyl carrier protein reductase (ENR) was used to select specific antibodies from a library of antibody fragments, single-chain Fv (scFv), displayed on filamentous phage. Analysis of the selected clones by BstNI fingerprinting and nucleotide sequencing showed that the scFv were derived from three different human VH germline genes. The binding specificities were confirmed by Western blots and ELISA. The scFv preparations reacted with B. napus ENR, but not with beta-keto reductase, nor enoyl reductase from Escherichia coli. Analysis of fragments generated by CNBr treatment indicates that the scFv 3.13 recognized an epitope located within the N-terminal 80 amino acids of the enzyme molecule. The scFv were used to detect ENR directly in extracts of B. napus seeds.
Ziegler
A
A
Scottish Crop Research Institute, Invergowrie, Dundee, United Kingdom. aziegl@scri.sari.ac.uk
Macintosh
S M
SM
Torrance
L
L
Simon
W
W
Slabas
A R
AR
D000213:Acyl Carrier Protein / Q000378:metabolism; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D015153:Blotting, Western; D001937:Brassica / Q000201:enzymology*; D003001:Cloning, Molecular; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D064429:Fatty Acid Synthases / Q000032:analysis / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000032:analysis* / Q000276:immunology*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment
D000213:Acyl Carrier Protein; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D064429:Fatty Acid Synthases