1.Basic information about proteins.
PAI1 (plasminogen activator inhibitor-1), also known as angiopoietin inhibitor-1, is an important blood coagulation regulatory protein. PAI1 plays a key role in thrombosis and the plasmin system.
2.Purpose of the experiment.
The recombinant PAI1 protein was constructed and expressed for subsequent study of the mechanism of PAI1 and physiological or pathological processes such as thrombolysis and angiogenesis. Catalog No. PA1000-5925
3.Design of Experiments.
Step 1: Gene cloning.
a.Primers were designed according to the PAI1 gene sequence in the GenBank database, and the PAI1 gene was amplified by PCR.
b.Ligation of the amplification product with a suitable vector (e.g., PET series) to construct a recombinant plasmid of the PAI1 gene.
Step 2: Validation of plasmid construction.
a.The recombinant plasmid DNA was extracted for restriction enzyme digestion verification.
b.The restriction enzyme digest products were independently identified and sequenced to verify the correctness of the recombinant plasmid.
Step 3: Transformation and expression of the recombinant plasmid.
a.Transformation of the recombinant plasmid into the expressing host (e.g., E. coli);
b.Host bacteria are cultured and PAI1 protein is expressed under appropriate induction conditions (e.g., IPTG induction);
c.SDS-PAGE and Western blot were used to detect protein expression.
Step 4: Protein purification.
a.Expression cells are collected, lysed, and cell lysates are prepared;
b.Pai1 was purified by affinity chromatography, ion exchange chromatography, gel chromatography and other methods.
Step 5: Identification of protein activity.
a.Fibrinolytic activity assay and enzyme-linked immunosorbent assay (ELISA) were used to detect the activity of recombinant PAI1.
b.Co-immunoprecipitation, mass spectrometry and other methods were used to verify the interaction between recombinant PAI1 and TPA and UPA.
4.Results and discussions.
The above experiments can successfully construct and express the PAI1 recombinant protein. The purified PAI1 recombinant protein can be obtained by protein purification and activity identification, and its ability to inhibit TPA and UPA activity can be verified. Further studies can use this protein to study the mechanism of PAI1 and physiological or pathological processes such as thrombolysis and angiogenesis.