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Production of a correctly assembled fibrinogen using transgenic silkworms

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Abstract

Fibrinogen from human blood is used as a main component of coagulants, including surgical tissue sealants. The development of a recombinant human fibrinogen (rFib) is anticipated to eliminate the risks of blood-borne infections. Here, we report the efficient production of rFib in a transgenic silkworm system. A silkworm line carrying cDNAs of the fibrinogen Aα and γ chains (Aα/γ-silkworm) produced Aα and γ chains in its cocoons, however, the Bβ chains were not detected from cocoons of another silkworm line carrying the cDNA of fibrinogen Bβ chains (Bβ-silkworm). A silkworm line for all three fibrinogen chains was generated by crossing Aα/γ-silkworms with Bβ-silkworms, which secreted Aα2Bβ2γ2 fibrinogen (rFib) into cocoons at high contents. The N-terminal amino acid sequences of the three rFib chains were identical to those of the corresponding chains of native fibrinogen (nFib). The N-glycan profile of the rFib comprised oligomannose-type (53%), complex-type (34%), and paucimannose-type (13%); neither high-mannose-type (six or more mannose residues) nor core-fucosylated glycans were observed. The coagulation activity of the rFib was evaluated for the amount of thrombin-released fibrinopeptide A (FpA) and the kinetics for turbidity increase (non-covalent network formation) in the solution. FpA release rates were equivalent between rFib and nFib; by contrast, the kinetics of the turbidity increase for rFib were accelerated nearly two-fold, for both the rate and maximum value, compared to those of nFib. These results demonstrate that the rFib produced in the transgenic silkworm system is comparable to nFib in both physical and coagulative properties. This rFib is a promising candidate component for safe hemostatic pharmaceuticals.

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Author notes

  1. Sachi Minagawa and Satoshi Sekiguchi have contributed equally to this work.

Authors and Affiliations

  1. Innovation Center, Nippon Flour Mills Co., Ltd., 5-1-3 Midorigaoka, Atsugi, Kanagawa, 243-0041, Japan

    Sachi Minagawa, Satoshi Sekiguchi & Yuzuru Nakaso

  2. Transgenic Silkworm Department, Immuno-Biological Laboratories Co., Ltd., 1-26-2 Asahi-cho, Maebashi, Gunma, 371-0014, Japan

    Takenori Igarashi & Masahiro Tomita

Authors
  1. Sachi Minagawa
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  2. Satoshi Sekiguchi
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  3. Yuzuru Nakaso
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  4. Takenori Igarashi
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  5. Masahiro Tomita
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Correspondence to Masahiro Tomita.

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Minagawa, S., Sekiguchi, S., Nakaso, Y. et al. Production of a correctly assembled fibrinogen using transgenic silkworms. Transgenic Res 29, 339–353 (2020). https://doi.org/10.1007/s11248-020-00202-1

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