Purification, Characterization And The Molecular Cloning Of The Fibrionlytic Enzyme From Eupolyphaga Sinesis And Tenebrio Molitor

Eupolyphaga sinesis belongs to Hexapeda, Blattaria, Corydiidae which broadly distributes in China (western) and Mongolia. The dried female body of ground beetle, which has been used as a kind of traditional Chinese medicine, contains variety of volatile oil, amino acid, protein, phenol, organic acid and alkaloid etc. Ground beetle as a kind of traditional Chinese medicine has the action to remove blood stasis with powerful effect, and to promote the healing of bone fracture. Some experiments of early stage showed that this drug inhibited liver cancer, stomach cancer and acute lymphocytic leukemia. The importance of this insect species in human diseases has increased interest in the science community and some government organizations of China.This paper established an improved fibrin plate method, which can be used for qualitative and semi-quantitative analysis. Several key factors were studied and discussed. The concentration of fibrinogen and plasminogen contained in the fibrin plate did not have any effect on the fibrinolytic area. However, the concentration of fibrinogen had influence on the observation and image gathering of fibrinolytic zones. It was difficult to clearly observe the fibrinolytic zones when the concentration of fibrinogen was below0.2mg/ml. The logarithm of the product of the two perpendicular diameters was linearly related to the logarithm of the concentration of urokinase when the concentration of urokinase was among2-250mU/μ1. In our experimental scheme, the optical concentration of urokinase was among10-20mU/μ1. We found that It was hard to denature plasmin by heating the agar-fibrin plate after the agar was solidified. However, the plasmin was completely denatured by heating at95℃for40min before the agar was solidified.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the most important and widely used technology which is mainly used to analyze the protein molecular weight. Fibrin zymography based on the SDS-PAGE is the best method for qualitative analysis of unknown fibrinolytic enzymes, especially for the analysis of molecular weight. In electrophoresis technique, molecular weight marker is the most important factor. However, it is difficult to detect protein molecular weight markers in fibrin zymography. In this study, some important factors, such as concentrations of fibrinogen and plasminogen, are discussed. Our results provide an efficient and convenient method which can clearly exhibit the dark blue bands of protein molecular weight (MW) markers and the transparent bands of fibrinolytic enzymes against the light blue background on one gel at the same time, and show high sensitivity.An eznyme with fibrinolytic activity was purified from ground beetle(Eupolyphaga sinesis) by ammonium sulfate precipitation between30%and70%saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and affinity chromatography on Benzamidine-Sepharose-6B. The purified fibrinolytic enzyme was designated as ESTL(Eupolyphaga sinesis trypsin-like enzyme) with molecular weight of22.8kDa by SDS-PAGE. The final purification degree was63.4%with a yield of9.7%and specific activity of1153U/mg.ESTL was only stable over a narrow range of pH values (6.0-10.5).The enzyme activity sharply decreased when the pH value was below4.0or above11.0. ESTL had much better stability at highly alkaline pH values comparing with highly acidic pH values. The maximal activity of ESTL was observed at pH9.5. ESTL was unstable at temperatures of40℃and above. Temperature optimum of ESTL was observed at45℃. The analysis of inhibitors showed that specific inhibitor of serine proteases (PMSF), trypsin inhibitors (TLCK, BPTI and benzamidine) inactivated ESTL almost completely, but specific inhibitor of cysteine proteases (E-64) and metalloprotease inhibitor (EDTA) had little effect on its activity (Fig.6A). These results indicate that ESTL are likely to be trypsin-like, not metalloprotease. The effect of metal ions on activity of ESTL showed a great difference. Na+, K+had little effect on the activity of ESTL over the tested range of concentration. Mg2+and Ca2+showed inhibition effect at high concentration. The amidolytic activity over Bz-Arg-pNA showed the hyperbolic concentration-velocity curve following the Michaelis-Menten kinetic model for the range of substrate concentrations analyzed.The gene of ESTL was cloned and the three-dimensional model of ESTL was constructed. The length of the cDNA is932bp, the open reading frame (ORF) has762bp. The ORF encodes354amino acids which contains a signal peptide with30amino acids. The molecular weight of ESTL is22.4kDa, and the isoelectric point is8.9. ESTL has the specific triad of serine proteinase, which contains the His41, Asp85and Ser179. The whole structure of ESTL consists of two parts and each one consists of one a-helix and six β-sheets. The two parts were connected by three coils, and the intermediate section between the two parts was the catalytic center with flexibility.Two proteins with fibrinolytic activity were partially purified from yellow mealworm (Tenebrio molitor) by ammonium sulfate precipitation between30%and70%saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and metal chelate on Cu-HiTrap-IMAC-FF, but the enzymes had not been completely separated from each other. The two partially purified fibrinolytic enzymes were designated as TMFE-I and TMFE-II (Tenebrio molitor fibrinolytic enzyme) with molecular weights of27.5kDa and24.9kDa by SDS-PAGE individually. The partially purified solution of TMTL-I and TMTL-II was considerably stable in the range of pH5-10and characterized by pH optimum of the enzymatic activity at8.0. Thermal stability of TMTL was excellent at45℃and below. The Km value was0.26mM for amidolysis of Bz-Arg-pNA. According to inhibitor analysis by fibrin plate method, phenylmethylsulfonyl fluoride (PMSF) and tosyl-lysine chloromethyl ketone (TLCK) inactivated TMTL almost completely, but trans-(epoxysuccinyl)-L-leucylamino-4-guanidinobutane (E-64) and EDTA had little effect on their fibrinolytic activity. According to metal ion analysis by fibrin plate method, the effect of metal ions on activity of TMTL showed a great difference. Na+, K+and Zn2+had little effect on the activity of TMTL. Mg2+and Cu2+showed inhibition effect on the fibrinolytic activity of TMTL, but Ca2+increased the fibrinolytic activity of TMTL at final concentration varying from0to30mM.The trypsin-like serine protease cDNA (TMTL) was cloned from yellow mealworm (Tenebrio molitor) and expressed in E. coli. The full-length cDNA of TMTL includes869bp (GenBank No. JN6624614) with the open reading frame of777bp and258coding amino acids. The protein TMTL with a molecular mass of26.77kDa has a trypsin-like serine protease catalytic triad, a unique trypsin initiation sites and expected binding sites of substrate active sits. The results of comparative analysis illustrated that the gene encoding of amino acid sequence was highly similar as those within a variety of insects. The3D model of TMTL was constructed based on the template of1pq7A. Under low temperature and low concentration of IPTG conditions, recombinant TMTL could not be expressed in the form of soluble enzyme in E. coli. The recombinant TMTL with6-His tag was purified and refolded using Ni+-HiTap affinity chromatography system from GE, following the manufacturer’s protocol. However, the enzyme activity of recombinant TMTL was much lower than the native TMTL purified from yellow mealworm. Compared with the native TMTL.