Expression and characterization of a synthetic protein C activator in Pichia pastoris

• Published in: Protein expression and purification 2002-12, Vol.26 (3), p.406-415
• Main Authors: Kunes, Yune Zhang, Sanz, Marı́a-Cruz, Tumanova, Irina, Birr, Cynthia A, Shi, Philip Q, Bruguera, Pau, Ruiz, Juan A, Sánchez-Martı́nez, Demetrio
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Fermentation; Genetic Engineering; Humans; Kinetics; Molecular Sequence Data; Oligopeptides - biosynthesis; Oligopeptides - genetics; Oligopeptides - metabolism; Pichia - genetics; Promoter Regions, Genetic - genetics; Protein C - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Snake Venoms - chemistry
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/S1046-5928(02)00560-0

Protein C activators are proteases that activate protein C in the mammalian coagulation system. A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway. We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence. This recombinant protein C activator was expressed in Pichia pastoris as a secreted glycoprotein (ILPCA) using the AOX1 promoter and the α-factor signal sequence. A fermentation protocol was developed that produced about 150 mg/L biologically active ILPCA secreted in the fermented broth. A two-step purification scheme was devised to purify ILPCA to ∼80% purity. The ILPCA produced has an apparent molecular weight of ∼68 kDa and a deglycosilated molecular weight of 28 kDa. Steady-state kinetic analysis reveals that ILPCA activates purified human protein C with a K m of 77 nM and a k cat of 0.39 s −1 . In conclusion, ILPCA is a recombinant protein that can be produced reliably and in large quantities under controlled manufacturing conditions, activates protein C, and can be used in coagulation assays as an alternative to native venom preparations.