Abstract
Agkisacucetin extracted from the venom of Agkistrodon acutus has been demonstrated to be a promising antithrombotic drug candidate in clinical studies due to its function as a novel platelet membrane glycoprotein (GP) Ib inhibitor. Agkisacucetin is a heterodimeric protein composed of α- and β-subunits with seven disulphide bonds. Both subunits form inactive homodimeric products, which cause difficulties for recombinant production. In this study, Agkisacucetin α- and β-subunits were inserted sequentially into the chromosome of Pichia pastoris at the mutant histidinol dehydrogenase gene and ribosomal DNA repeat sites, respectively. By optimizing the gene copies and productivity of each subunit by drug screening, we successfully obtained a recombinant strain with balanced expression of the two subunits. Using this strain, a yield greater than 100 mg/L recombinant Agkisacucetin in fed-batch fermentation was reached. The recombinant Agkisacucetin possessed extremely similar binding affinity to recombinant GPIb and human platelets in in vitro assays, and its ristocetin-induced platelet aggregation activity ex vivo was identical to that of the extracted native Agkisacucetin, demonstrating that the yeast-derived Agkisacucetin could be an effective alternative to native Agkisacucetin. Moreover, this study provides an effective strategy for balancing the expression and production of heterodimeric proteins in P. pastoris.
Highlights
Cardio-cerebral vascular diseases, thrombosis, remain the most serious life-threatening diseases[1]
This study is the first report of the successful establishment of a balanced expression strain and pilot-scale production of functionally active rAgkisacutacin in P. pastoris
A common challenge for the expression of heterodimeric proteins is that each subunit in a heterodimeric protein can form homodimers, which could act as a decoy for its active partners and interfere with the production rate and with downstream purification[15]
Summary
Cardio-cerebral vascular diseases, thrombosis, remain the most serious life-threatening diseases[1]. GPIb is one of the key factors that mediate platelet adhesion under high shear conditions and an important target for new antiplatelet drug development[2]. Agkisacucetin, which is a C-type-like lectin protein (CLP), binds to GPIb to prevent its binding to vWF, inhibiting platelet adhesion and aggregation[6,7]. Agkisacucetin did not significantly cause platelet activation and bleeding in murine tests, which, together with other evidence, suggests that it has additional effects beyond its inhibitory role in the GPIb-vWF interaction[4]. An oxidized sulphhydryl group is present at the N-terminus of the β -subunit in Agkisacucetin[7] This native structure causes unusual difficulties for recombinant production, for obtaining high-yield biologically active products in large-scale production. This study established an effective strategy for balancing the expression and production of rAgkisacucetin, which could be employed for the production of other heteromultimeric proteins
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