Cloning and expression of the novel gene -: PV242 of earthworm fibrinolytic enzyme

One kind of protein from earthworm (Lumbricus bimastus) with the molecular mass about 30 ku was extracted by SDS-PAGE. Then the N-terminal of the protein was sequenced. The PCR primer was designed according to the N-terminal sequence, and its cDNA fragment was obtained by RT-PCR. The cDNA was cloned into pGEMT-vector and sequenced. The sequence showed that the fragment was 888 bp, with the ORF of 726 bp and the 3' untranslation terminal area. The ORF encoded a protein of 242 amino acids, so the protein was named as PV242. The prediction of protein structure shows that the PV242 has two domains, between which is the active sites-His44 and Ser191. At the same time, the pI 4.33 of PV242 was detected, and it is a protease in trypsin family of serine protease superfamily. PV242 was expressed in pTrxFUS expression system as the fusion protein TrxA-PV242 in soluble form, the TrxA was a molecular chaperon which made the expressed protein soluble. The fusion protein was purified by ion exchange chromatography. The purified fusion protein TrxA-PV242 has fibrinolytic activity.