Synthesis of a Gene Encoding Fibrinolytic Enzyme from Earthworm and Its Expression by Pichia pastoris

By selecting the biased codon in P.pastoris,the new gene EFE-3D encoding earthworm fibrinolytic enzyme,consisting of 748 bp,was divided into 32 oligonucleotide fragments.The 32 synthetic fragments synthesized in vitro were assembled into one complete target fragment with only one step by a PCR approach and was cloned into pPIC9K plasmid vector.The expressing plasmid containing the synthetic gene was introduced into Pichia pastoris genome by electroporation.The secreted product was detected by Western Blotting.Fibrinolytic activity of product EFE-3D on fibrin plate was(3 500) mm~2/mL,thus the activity in 1 L fermentation broth was equivalent to 48 mg EFE activity.