Cloning and expression of earthworm fibrinolytic enzyme PM246 in Pichia pastoris

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 × 106 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.