We have cloned, expressed, and purified a novel earthworm fibrinolytic enzyme (EFE) of Lumbricus rubellus in Pichia pastoris. Its cDNA sequence (GenBank Accession No. AY178854) revealed a 747 bp region containing an intact ORF that encodes a protein of 246 amino acid residues, designated as EFE PM246. While EFE PM246 is distinct, its cDNA shows a high degree of sequence homologies with four other EFE cDNAs registered in GenBank. The recombinant EFE PM246 was active, showing a fibrinolytic activity of 7.5 x 10(6) U/L in basal salts medium, a higher fibrinolytic activity than those produced in other expression systems. The recombinant EFE PM246 expressed in basal salts medium was purified by a three-step purification procedure with a recovery rate of about 20%. This is the first report detailing the successful purification of a genetically engineered earthworm fibrinolytic enzyme. The main physiochemical features of the EFE PM246, including temperature stability, pH resistance, and sensitivity to some protein inhibitors, were also characterized. (c) 2005 Elsevier Inc. All rights reserved.