Expression of Recombinant Tissue Plasminogen Activator - Plasminogen Activator Inhibitor Complex in Mammal Cells as Quality Control Materials in Immunoassay.

Abstract

Tissue plasminogen activator - plasminogen activator inhibitor complex (tPAIc) is a critical biomarker to assess fibrinolytic dysfunction, which is widely used in clinics. Quality control material (QCM) plays an important role in immunoassays for human tPAIc. The QCM of tPAIc are derived from human plasma with many disadvantages. Recombinant protein is a promising substitute for human plasma to work as the source of QCM. However, tPAIc is a protein complex, consisting of three parts, tPA-A, tPA-B, and PAI-1, which makes the expression more difficult. This study aimed to obtain recombinant tPAIc QCM with excellent performance for immunoassay. Three recombinant plasmids that matched each part of the protein complex were constructed and co-transfected to HEK293F cells. The optimal molar ratio of three plasmids was further explored. Each part of the proteins was secreted from cells and the target protein tPAIc was self-assembled in the supernatant. After being identified by western blot and chemiluminescent immunoassay (CLIA), calculating the concentration of tPAIc in the supernatant, tPAIc was diluted to approximately 50 ng/mL and 5 ng/mL, distributed, and lyophilized in ampoules, working as QCM in tPAIc immunoassay. The homogeneity, stability, and recovery of the QCM were further evaluated. The three plasmids were successfully constructed. The target protein complex, tPAIc, was obtained in the supernatant at about 6500 ng/mL, under the best three plasmids co-transfected molar ratio 1:1:1. The QCMs were uniform in different ampoules. They were verified to be highly stable for at least 1 year when stored at 4 ℃ and - 20 ℃. The recombinant tPAIc QCMs for immunoassay were obtained with high quality to replace plasma-derived QCMs, which provides valuable insight into more application scenarios of recombinant proteins.