A Study of the Effect of Human Prothrombin’s Signal Peptide on the Secretion Efficiency of Recombinant Human FIX in the HEK۲۹۳T Cell Line

Objective: Eukaryotic proteins generally have signal peptides which are not only crucial for their secretion efficiencies but are important for their expression levels. Coagulation factor IX (FIX) is a glycoprotein that plays a fundamental role in the blood coagulation pathway. Reduced levels or dysfunctional FIX are associated with hemophilia B. This study investigates the function of the human prothrombin signal peptide in an attempt to improve the human FIX (hFIX) secretion efficiency in a heterologous expression system. With this aim, we have used the SignalP and PrediSi programs for in silico evaluation of the signal peptide efficiency prior to conducting this experiment. Methods: We used molecular techniques to amplify and join the coding region of the human prothrombin signal peptide to the cDNA of mature hFIX. The chimeric fragment was examined for transient expression in a mammalian cell line (HEK۲۹۳T) in comparison with the native hFIX, under a CMVp regulation. Using the neural network-based prediction programs, we evaluated the scores for cleavage position and secretion efficiency of the human prothrombin and hFIX signal peptides. The expression efficiencies of hFIX expressed by the recombinant cells were analyzed by RT-PCR and ELISA. Results: In silico analysis more efficiently predicted the human prothrombin signal peptide with a high score compared to the native hFIX signal peptide. This data was confirmed by the RT-PCR and ELISA results obtained from expression analyses at the RNA and protein levels, respectively. Conclusion: The present study showed that the signal peptide derived from the human prothrombin has the potential for efficient secretion of hFIX as evidenced by the results taken from a transient expression system. The results were consistent with in silico analysis. This replacement could be evaluated in a stable state condition.

Keywords:

Human factor IX , Prothrombin , Secretion efficiency , Signal peptide , فاکتور ۹ انسانی , پروترومبین , کارآیی ترشح , پپتید نشانه