Cloning of Lumbrokinase from Earthworm and Its Expression in Pichia pastoris GS115

We have cloned and expressed a novel earthworm fibrinolytic enzyme (EFE) of Lumbricus rubellus in Pichia pastoris. Its cDNA sequence (GenBank Accession No. DQ202401) revealed a 738 bp region containing an intact ORF that encodes a protein of 245 amino acid residues, containing a signal peptide of 7 amino acid residues and a mature peptide of 238 amino acid residues, designated as EFE F238. Its cDNA shows a high degree of sequence homologies with four other EFE cDNAs registered in GenBank. The gene encoding the native form of F238, with a 5´ non-functional end removed, was obtained by PCR amplification and was sub-cloned into pPIC9, a yeast expression and secretion vector. SDS-PAGE analyses showed that EFE F238 could be secreted into the culture medium. The artificial fibrin plate experiment demonstrated that EFE F238 was active, showing a fibrinolytic activity of about 130 urokinase units/mg protein in basal salts medium.