Expression, Purification and Characterization of Recombinant Human Coagulation Factor XIIIa in Pichia Pastoris

Abstract

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.

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“…It is known that the experimental conditions are important for protein expression in this yeast. Unlike Cheng et al (2021) who achieved expression of human coagulation factor XIIIa at 30°C with 1% methanol every 24h for 120h, Wang et al (2015) induced expression of camel chymosin at pH 4.07 and even at lower temperature of 28°C after 8h. After evaluation of the different parameters, expression of human serum albumin was performed at 28°C for 24 hours in an acidic pH of 5.75 and the methanol concentration was set between 0.5 and 2% and for every 2 hours (Lau et al, 2012).…”

“…Human Coagulation Factor XIIIa, which was insoluble after its complicated expression in E. coli (Nikolajsen et al, 2014) was successfully produced by Chang et al, (2011) using P. pastoris. As used in the present study, these authors also employed the methanol inducible AOX1 promoter to efficiently express the protein for which they evaluated different biological activities (Cheng et al, 2021). The same AOX1 promoter controls the expression of the human camel chymosin in P. pastoris (Wang et al, 2015).…”