Expression of recombinant rabbit tissue factor in Pichia pastoris, and its application in a prothrombin time reagent.
Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains. It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation. The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies. To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris. Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity. A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility.
Brucato
Cheryl L
CL
Research and Development, Instrumentation Laboratory, 113 Hartwell Ave, Lexington, MA 02421, USA.
Birr
Cynthia A
CA
Bruguera
Pau
P
Ruiz
Juan A
JA
Sánchez-Martínez
Demetrio
D
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005167:Factor VII / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D015870:Gene Expression; D010843:Pichia / Q000235:genetics*; D011517:Prothrombin Time; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D013925:Thromboplastin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa