Cloning of thrombolytic enzyme (lumbrokinase) from earthworm and its expression in the yeast Pichia pastoris

Lumbrokinase (PI239, GenBank Accession No. AF433650) from the earthworm Lumbricus bimastus has been identified. The cDNA of PI239 is composed of 852bp and includes an open reading frame that encodes two parts of the protein: a signal peptide of 44 amino acids and a mature peptide of 239 residues. The cDNA of PI239 exhibits a high degree of sequence identity with other lumbrokinase genes, ranging from 87.6% (F-III-I) to 98.3% (EFE-3). The gene encoding the native form of PI239, with a 5' non-functional end removed, was obtained by PCR amplification and was sub-cloned into pPICZalpha-A, a yeast expression and secretion vector. SDS-PAGE and Western blot analyses showed that rPI239 secreted into the culture medium was specifically recognized by the wild type lumbrokinase polyclonal antibody and was able to dissolve artificial fibrin plates.