MOLECULAR-CLONING AND NUCLEOTIDE-SEQUENCE OF CDNA-ENCODING RAT-KIDNEYLONG-CHAIN L-2-HYDROXY ACID OXIDASE - EXPRESSION OF THE CATALYTICALLYACTIVE RECOMBINANT PROTEIN AS A CHIMERA

Long-chain L-alpha-hydroxy acid oxidase from rat kidney is a member of the family of FMN-dependent alpha-hydroxy-acid-oxidizing enzymes. Wit h the knowledge of the recently determined amino acid sequence, the cD NA encoding the enzyme has now been cloned using the polymerase chain reaction. The 1648-bp cDNA contains an open reading frame coding for t he 352 residues of the previously determined sequence, preceded by a m ethionine codon. In addition, several clones were found to present a n ine-base insertion, predicting the existence of an isoform with a trip eptide VRK inserted between residues 188 and 189 of the mature protein . The presence of about 10% of this isoform in the oxidase purified fr om rat kidney was indeed identified by amino acid sequencing. A recomb inant active enzyme was obtained as a protein fused to glutathione S-t ransferase using the bacterial expression plasmid pGEX-3X. Physico-che mical characterization indicated, for the fused enzyme, properties sim ilar to those of the rat kidney protein. When the chimaera was submitt ed to factor Xa, proteolysis at the engineered cleavage point was poor . Separation of hydroxy acid oxidase from glutathione S-transferase co uld not be achieved with trypsin either. With both proteases, the init ial cleavage point appeared to be in a peptide loop internal to the hy droxy acid oxidase sequence, close to or in the tripeptide insertion l ocus and not at the engineered factor-Xa-cleavage point. Comparative t ryptic proteolysis of the rat kidney enzyme yielded a form cleaved in the same loop.