Anti-peptide immunoglobulins from rabbit and chicken eggs recognise recombinant human dihydroorotate dehydrogenase and a 44-kDa protein from rat liver mitochondria.
Mitochondrially bound dihydroorotate dehydrogenase catalyses the fourth sequential step in the de novo synthesis of uridine monophosphate. 312-bp and 983-bp regions of the human dihydroorotate dehydrogenase sequence (1496 bp) were amplified by the polymerase chain reaction, and subcloned into the expression vector pQE 32. The identity of the PCR products was verified by dideoxynucleotide sequencing. Transformation of Escherichia coli strain M15 resulted in expression of 13-kDa and 36-kDa proteins with an affinity tag consisting of six consecutive histidine residues; these proteins could be purified by solubilisation in 8 M urea and by chromatography on a Ni2+-chelating resin. In immunoblotting analyses, the fusion proteins were recognised by polyclonal avian and mammalian anti-peptide immunoglobulins. These were generated against synthetic peptides corresponding to two amino acid sequences deduced from human and rat cDNA of dihydroorotate dehydrogenase. The peptides were synthesized as multiple copies on a branching lysyl matrix. Rabbits and laying hens were immunized with these peptides without conjugation to a carrier protein. Comparison of the anti-peptide immunoglobulins produced from egg yolk and rabbit serum demonstrated that avian anti-(dihydroorotate dehydrogenase) immunoglobulins may be considered a superior alternative to the mammalian equivalent; antibodies from both sources were applicable for all immunochemical purposes. Here, these antibodies were applied for identification of a 44-kDa protein from rat liver mitochondria, which was correlated with dihydroorotate dehydrogenase activity.
Knecht
W
W
Institute for Physiological Chemistry, School of Medicine, Philipps-University, Marburg, Germany.
Köhler
R
R
Minét
M
M
Löffler
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D000090722:Dihydroorotate Dehydrogenase; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007136:Immunoglobulins / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000276:immunology*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010455:Peptides / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins
D000090722:Dihydroorotate Dehydrogenase; C016512:IgY; D007074:Immunoglobulin G; D007136:Immunoglobulins; D010455:Peptides; D011994:Recombinant Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors