Expression and purification of recombinant serine protease domain of human coagulation factor XII in Pichia pastoris

Abstract

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human βFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.

Advances in Metabolic Engineering of Pichia pastoris Strains as Powerful Cell Factories

“…Human coagulation factor XII plays an important role in thrombosis, and its abundant supply is necessary for inhibitor screening in the development of antithrombotic drugs. The recombinant serine protease domain of human coagulation factor XII has been expressed in P. pastoris X-33 with a yield of 20 mg/L and a clotting activity similar to that of its natural counterpart [110]. Compared with these proteins which can be expressed easily in the soluble form, high-level microbial expression of human membrane proteins can be a real challenge.…”

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

“…This technique has been used for more than 20 years and is highly suitable for many bodily proteins, with approximately 40% being replenished into soluble and biologically active forms [ 138 , 149 ]. In this process, the inclusion bodies are denatured with a denaturing buffer containing either 6 M guanidine hydrochloride (GdnHCl), 8 M urea, or 0.3% sarkosyl (n-lauroyl sacosinate) [ 150 ]. However, in most situations, a substantial amount of precipitation occurs during the refolding of the proteins, resulting in a major reduction in the total yield of the desired proteins.…”