EXPRESSION OF THE PEANUT PEROXIDASE CDNA IN ESCHERICHIA-COLI AND PURIFICATION OF THE PROTEIN BY NICKEL AFFINITY

A cDNA sequence containing the entire cationic peanut (Arachis hypogae a) peroxidase (EC 1.11.1.7, CPRX) coding sequence, including a 22-amin o acid signal peptide, was extended using a synthetic oligonucleotide with a six histidine-tag at the C-terminal and expressed in bacteria. The expressed protein was purified on a Ni2+-nitrilotriacetic acid (NT A) column and the resulting protein had a molecular mass corresponding to an unglycosylated polypeptide. Western blot analysis showed the pr otein to be recognized by polyclonal antibodies raised against native CPRX and monoclonal antibodies specifically recognizing six histidine- tag, but not by monoclonal antibody which recognizes carbohydrate-cont aining epitopes of CPRX. (C) Elsevier, Paris.