Construction And Functional Characterization Of Human Coagulation Factor ?,Primary Exploration Of Two Novel Anticoagulants Binding Sites On Human Serum Albumin

Objective: To express the active Factor-XII catalytic domain?FXII-SPD?by the Pichia pastoris system and analysis of the r FXII-SPD Function.Methods: First,the c DNA of the target protein?coagulation factor XII Serine protease domain?was synthesized and cloned into the p PICZ?A plasmid vector by Xho I and Sal I double-cleavage sites,and electroporated into the competent P.pastoris X33 for expression.The expressed recombinant protein was purified by nickel ion metal chelate affinity chromatograp Hy,and then further purified by size exclusion chromatograp Hy.The expression of the target protein was identified by Western blotting and mass spectrometry,Subsequently,the Michaelis constant of its specific chromogenic substrate S-2302 was determined to analyze the activity of the target protein,and its effect on the blood coagulation process was examined.At the same time test its effect on the blood coagulation processResults: The expressed recombinant protein was purified by nickel ion metal chelate affinity chromatography and further purified by molecular exclusion chromatography.Finally,the active protein with good homogeneity and purity of up to 98%,What more,The expression amount could up to 20 mg/L.Meanwhile it can accelerate the formation of clots in the plasma.Conclusion: 1.The recombinant FXII-SPD protein was successfully expressed in the Pichia pastoris system.2.The expressed recombinant FXII-SPD protein was successfully purified using Ni 2+-NTA affinity chromatography and gel filtration.3.The recombinant FXII-SPD protein has an amidolytic activity in vitro and promotes coagulation.Objective: Growth of the human serum albumin-new anticoagulant drug complex crystals,and analysis of specific binding site or interaction mechanism.Methods: 1.Preparation of HSA-new anticoagulant drug complex and growth of HSA-new anticoagclant drug complex crystal.2.growth of the HSA crystal,HSA crystal was immersed in a bath containing a novel anticoagulant drug which may be diffused into the crystal of the HSA to form a HSAnew anticoagulant drug complex crystal.3.HSA-new anticoagulant drug binary complex crystal was sent to Shanghai Synchrotron Radiation Center for X-ray diffraction experiment.4.Analysis of the crystal structure of HSA-new anticoagulant drug complex:?1?Model construction: After obtaining the original crystal data,we will use the molrep program of CCP4 to use the HSA crystal structure in the PDB database as a template,and use the fast rotation and translation functions to find the correct atom model.?2?Structural correction: After obtaining the appropriate model,we will use Refmac and CNS and the p Henix program to correct it and determine the location of the anticoagulant.?3?Structural refinement: Once the electron density map and the appropriate coordinate file are obtained,the structure will refined by the Coot and Py MOL programs.Results: 1.Crystallization conditions of human serum albumin-argatroban complex: 31%³5% PEG3350;p H 7.4 PB;H₂O.2.Crystallization conditions of human serum albumin-rivaroxaban complex: 31%³5% PEG3350;p H 7.4 PB;H₂O.3.The structural data of human serum albumin-argatroban and human serum albuminrivaroxaban binary complex eutectic have been collected,It was found that some of the amino acids in human serum albumin may be rivaroxaban and argatroban electronic cloud density.Conclusion: Analyzing of the complex crystal structure data,it was found that argatroban and rivaroxaban may be bind to human serum albumin.