Challenges of expressing recombinant human tissue factor as a secreted protein in Pichia pastoris. Issue 9 (3rd October 2022)

• Record Type: Journal Article
• Authors: Jalili-Nik, Mohammad; Soukhtanloo, Mohammad; Mojarrad, Majid; Sadeghian, Mohammad Hadi; Mashkani, Baratali
• Is Part Of: Preparative biochemistry & biotechnology. Volume 52:Issue 9(2022)
• Page Start: 1001
• Page End: 1007
• Publication Date: 2022-10-03
• Subjects: α-factor -- Pichia pastoris -- pPICZ-αA -- tissue factor; Biochemistry -- Technique -- Periodicals; Biotechnology -- Technique -- Periodicals; Biochemistry -- methods -- Periodicals; Biotechnology -- methods -- Periodicals; 660.6
• DOI: 10.1080/10826068.2021.2023823
• Languages: English
• ISSNs: 1082-6068

Abstract

Abstract: Tissue factor (TF) is the core reagent in the prothrombin time (PT) assay. In this study, expression and α-factor mediated secretion of three forms of tissue factor (full-length TF (Full-TF), extracellular plus transmembrane domain (TED-TF), and only extracellular domain (ED-TF) were investigated in Pichia pastoris . The amino acid sequence of TF was obtained from the UniProt database, back-translated and codon-optimized for expression in Pichia pastoris . The Full-TF sequence was synthesized but the ED-TF, TED-TF coding fragments were extracted from the Full-TF by PCR. All the coding sequences were cloned into pPICZαA vector in-frame with the α-factor; and electroporated into KM71H. The culture supernatants and the cell lysates were analyzed using SDS-PAGE, dot-blotting, and Western-blotting for expression of TF. The Full-TF and TED-TF expression vector pPICZαA were successfully inserted into the KM71H, but the product was not detected in the SDS-PAGE analysis of the culture supernatant. However, ED-TF expression and secretion was verified by SDS-PAGE, dot blotting, and Western blotting. It seems that the TM domain in the Full-TF and TED-TF have an important role in impairing α-factor-mediated secretion of TF. Therefore, further investigation is necessary to overcome challenges of expressing Full-TF as a heterologous protein in P. pastoris .