Background: Coagulation factor XIIIa(FXIIIa) plays a critical role in the final stage of blood coagulation. It is extremely important in wound healing, tissue repairing and promoting cell adhesion. The deficiency of the coagulation factor can cause hemorrhage and slow wound healing.
Objective: In this study, recombinant pPICZαC-FXIIIa was expressed in Pichia pastoris, purified as well as its biological activity was determined.
Methods: The FXIIIa fragment obtained from the human placenta was inserted into pPICZαC to obtain pPICZαC-FXIIIa, which was transformed into X33 after linearization, and FXIIIa inserted into Pichia pastoris X33 was screened for methanol induction. The expressed product was identified by western blotting, then the supernatant was purified by affinity chromatography, and the purified product was determined by plasma coagulation experiment.
Results: Polymerase Chain Reaction(PCR) showed that the FXIIIa fragment of 2250 bp was inserted successfully into pPICZαC. The expression and purification products of the same molecular weight as target protein(about 83 kDa) were obtained, which solidified significantly when reacted with plasma.
Conclusion: The expression and purification products were successful, with sufficient biological activity, which can be used as a candidate FXIIIa hemostatic agent in genetic engineering.
Keywords: pPICZαC-FXIIIa, Pichia pastoris, secretory expression, protein purification, biological activity, hemostatic agent.
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