Purification, cloning, and expression of human plasma hyaluronidase

Hyaluronidase was purified from human plasma using Triton X-114 phase extractions and ion-exchange chromatography. Monoclonal antibodies generated against the purified protein by a novel screening assay mere utilized to isolate homogeneous enzyme fear microsequencing, The amino acid sequences obtained matched a cDNA in the Expressed Sequence Tag database which, with 5'-RACE-PCR, was used to alone the plasma hyaluronidase gene, termed Hgal-1. Hyal-1 codes for a protein of 435 amino acids that is over 40% identical to PH-20, sperm-specific hyaluronidase. Unlike PH-20, which is only expressed in testis, transcripts of Hyal-1 were found in multiple tissues, Hyal-1 stably expressed in human embryonic kidney cells resulted in a 3,000 fold increase of secreted immunoreactive hyaluronidase activity that was biochemically indistinguishable from human plasma hyaluronidase, By immunological, molecular and biochemical criteria, me conclude that Hyal-1 is the predominant hyaluronidase found in human plasma. (C) 1997 Academic Press.