Cloning And Expression Of Fibrinolytic Enzyme From Two Speices Of Arthropod

Fibrinolytic enzyme in the same category of the trypsin-like serine proteases are very important alkaline proteases with a stronger degradable fibrinolytic activity. They were prevalent existed in anaimal, plant and fungus. And many fibrionolytic enzyme have been used clinically as thrombolytic agent. In this paper, we cloned a fibrionlytic enzyme from scorpion and expressed a fibrinolytic enzyme of tenebrio molitor in E.coli BL21(DE3) which has fibrinolytic activities.We designed two primers based on a relatively conservative amino sequence of fibrinolytic proteases of other species. And we got a gene segment by degenerate oligonucleotide primer PCR. Then we got the3’ fragment and5’fragment of the fibrinolytic enzyme by RACE technique. Then a fragment of1427bp representing the full-length cDNA sequence of the enzyme was obtained by sequence assembly of the above sequences. The full-length of STSP cDNA is1427bp, containing a single open reading frame (ORF) of1176bp that encodes a protein of391amino acids. Based on the analysis of BLAST of NCBI, the enzyme amino sequence showed an identity of30%to45%with the fibrinolytic trypsin-like serine protease of other species. There is a signal peptide with18amino acid residues in N-ternimal of the enzyme, and the molecular weight of the enzyme is42kD. From the analysis of biological software, the enzyme has the typical structure of catalytic triad. We also constructed the three-dimensional model structure of fibrinolytic enzyme of scorpion. Based on the structure, there is no a-helix nor β-sheet in both substrate binding sites and active sites which indicates the area can be easily distorted and promotes the binding with substances. And it also demonstrated the enzyme was conformed to the mechanism of induced fit.We used pET32a+plasmid to construct the expression vectors, and had succeeded expressed the fibrinolytic enzyme of tenebrio molitor in E.coli BL21(DE3). The optimal cuLture conditions of pET32a+-CS were determined as following:cultivation at30℃in LB medium, induction at middle stage of exponential growth with0.4mM IPTG, and post-induction expression for5h. In this condition, we couLd get the high level of soluble the interest protein, although most expressed protein existed in a form of inclusion bodies. We used urea to denature the inclusion body, and then the denatured inclusion bodies were purified by GE HiTrap IMAC FF with Ni-NTAchelate agarose. At last, the purified denatured protein was dialyzed with a gradient decrease urea buffer for renaturation. And we got highly purified expressed enzyme, which have fibrinolytic activity. Besides, we also simulated the three-dimensional model of the fibrinolytic enzyme of tenebrio molitor.