Partial amino acid sequence of porcine 1,25-dihydroxyvitamin D3 receptor isolated by immunoaffinity chromatography

Monoclonal antibodies against the porcine 1,25-dihydroxyvitamin D3 receptor were immobilized on Sepharose CL-4B and used to obtain a highly purified 1,25-dihydroxyvitamin D3 receptor fraction with a 45% recovery of the 1,25-dihydroxyvitamin D3 binding capacity. The porcine receptor was purified to homogeneity by preparative electrophoresis and digested in sodium dodecyl sulfate/polyacrylamide gels with Staphylococcus aureus strain V8 protease. The resulting peptides were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, electrophoretically transferred to polyvinylidene difluoride membranes, and directly sequenced. The generation and isolation of peptides by this method allows sequencing of proteins present in trace amounts as well as those whose amino termini have been modified. The 1,25-dihydroxyvitamin D3 receptor amino acid sequence corresponded to the sequence predicted from a recently cloned receptor cDNA obtained from rat kidney mRNAs.