The Expression Of Treponema Pallidum Metalloprotease With The Study Of Its Fibrinolytic Potential

Objective: To investigate the fibrinolytic potential and its mechanism of Treponema pallidum metalloprotease Tp0751, providing the experimental basis for further development of syphilis pathogenic mechanism.Methods:(1) Expression and purification of recombinant Tp0751: The Tp0751 without sequence of signal peptide were obtained in Genbank, to construct the prokaryotic expression vector pET30a(+)/Tp0751, pET30a(+) /Tp0751 H198 A and pET30a(+)/Tp0751 H202 A, and transformed into E.coli BL21 for Tp0751 protein expression after identified by double enzyme digestion, PCR and gene sequencing. Recombinant Tp0751(rTp0751) purified with Ni-NTA affinity chromatography. Protein concentration was tested by BCA kit. rTp0751 were determined by Western blot and SDS- PAGE to identify it’s purity.(2) In vitro fibrinogen degradation assays of wild type Tp0751: Using recombinant wild type Tp0751 to incubate with plasminogen-free human fibrinogen(the final quality of target protein is 30 μg, the final quality of Fg is 60 μg, the total volume is 100μL), samples were removed at hourly intervals and the fibrinogenolytic activity was determined by SDS-PAGE analyses.(3) In vitro fibrinogen degradation assays of H198 A mutant Tp0751 : Using recombinant H198 A mutant Tp0751 to incubate with plasminogen-free human fibrinogen(the final quality of target protein is 30 μg, the final quality of Fg is 60 μg, the total volume is 100μL), samples were removed at hourly intervals and the fibrinogenolytic activity was determined by SDS-PAGE analyses.(4) In vitro fibrinogen degradation assays of H202 A mutant Tp0751:Using recombinant H202 A mutant Tp0751 to incubate with plasminogen-free human fibrinogen(the final quality of target protein is 30 μg, the final quality of Fg is 60 μg, the total volume is 100μL), samples were removed at hourly intervals and the fibrinogenolytic activity was determined by SDS-PAGE analyses.Results:(1) Expression and purification of recombinant Tp0751: It is confirmed that the inserted gene sequence of recombinant plasmid pET30a(+)/Tp0751 was the same sequence to that sequence logined on GenBank. Product of that sequence is 26kDa-size which can solubly expressed, the purity of the recombinant protein is about 90%. According to the Western-blot result, only syphilis sera were active with rTp0751 specifically.(2) In vitro fibrinogen degradation assays of wild type Tp0751: The wild type Tp0751 can degradate plasminogen-free human fibrinogen which absolutely degradate it 24 h post-incubation.(3) In vitro fibrinogen degradation assays of H198 A mutant Tp0751:The H198 A mutant Tp0751 can also degradate plasminogen-free human fibrinogen which partly degradate it 24 h post-incubation.(4)In vitro fibrinogen degradation assays of H202 A mutant Tp0751 : The H202 A mutant Tp0751 mainly can not degradate plasminogen-free human fibrinogen which hardly degradate it 24 h post-incubation.Conclusions:(1) The wild type Tp0751 can absolutely degradate plasminogen-free human fibrinogen;(2) The HEXXH motif may have an important role in Tp0751 to the degradation of plasminogen- free human fibrinogen; residue H202, but not H198, from the pallilysin HEXXH motif is required for zinc coordination.