title: Balancing the Expression and Production of a Heterodimeric Protein: Recombinant Agkisacutacin as a Novel Antithrombotic Drug Candidate.
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.
authors.affiliation: 1] The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China [2] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [3] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Yugang
authors.initials: Y
authors.lastname: Guo
authors.affiliation: 1] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [2] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Jia
authors.affiliation: 1] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [2] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: 1] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [2] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Changsheng
authors.initials: C
authors.lastname: Shao
authors.affiliation: 1] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [2] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.affiliation: 1] The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China [2] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [3] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Jiajia
authors.initials: J
authors.lastname: Ma
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Fang
authors.affiliation: The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.affiliation: 1] The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China [2] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [3] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.affiliation: Zhaoke Pharmaceutical (Hefei) Co. Ltd., Hefei, Anhui, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Qian
authors.affiliation: Zhaoke Pharmaceutical (Hefei) Co. Ltd., Hefei, Anhui, China.
authors.forename: Xiangrong
authors.initials: X
authors.lastname: Dai
authors.affiliation: Zhaoke Pharmaceutical (Hefei) Co. Ltd., Hefei, Anhui, China.
authors.forename: Guohui
authors.initials: G
authors.lastname: Zhang
authors.affiliation: 1] The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China [2] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [3] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Tian
authors.affiliation: Lee's Pharmaceutical Holdings Limited, Shatin, Hong Kong, China.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Xiaoyi Li
authors.affiliation: 1] The CAS Key Laboratory of Innate Immunity and Chronic Disease, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China [2] Hefei National Laboratory for Physical Sciences at Microscale, Engineering Technology Research Center of Biotechnology Drugs, Anhui Province, University of Science and Technology of China, Hefei, China [3] Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology, Institute of advanced technology, University of Science and Technology of China, Hefei, China.
authors.forename: Weihua
authors.initials: W
authors.lastname: Xiao
mesh_terms: D017836:Agkistrodon / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D061249:Batch Cell Culture Techniques; D003435:Crotalid Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012310:Ristocetin / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D003435:Crotalid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D021122:Protein Subunits; D011994:Recombinant Proteins; C401844:agkisacutacin; D012310:Ristocetin