Expression and characterization of a synthetic protein C activator in Pichia pastoris.
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 alpha-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 approximately 80% purity. The ILPCA produced has an apparent molecular weight of approximately 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.
Kunes
Yune Zhang
YZ
Research and Development, Instrumentation Laboratory, 113 Hartwell Ave., Lexington, MA 02421, USA.
Sanz
María-Cruz
MC
Tumanova
Irina
I
Birr
Cynthia A
CA
Shi
Philip Q
PQ
Bruguera
Pau
P
Ruiz
Juan A
JA
Sánchez-Martínez
Demetrio
D
D000818:Animals; D001483:Base Sequence; D005285:Fermentation; D005818:Genetic Engineering; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011486:Protein C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D012910:Snake Venoms / Q000737:chemistry
D009842:Oligopeptides; D011486:Protein C; D011994:Recombinant Proteins; D012910:Snake Venoms; C047135:protein C activator peptide