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Abstract <p>One of the most significant bacteriophage technologies is phage display, in which heterologous peptides are exhibited on the virion surface. This work describes the display of λ decorative protein D<sub>λ</sub> linked to the E protein domain III of Zika virus (D<sub>λ</sub>-ZE<sub>DIII</sub>), to the GFP protein (D<sub>λ</sub>-GFP), or to different domain III epitopes of the E<sub>ZIKV</sub> protein (D<sub>λ</sub>-TD), exhibited on the surface of an in vitro evolved lambda phage (λ<sub>evo</sub>). This phage harbors a gene <i>D</i> deletion and was subjected to directed evolution using <i>Escherichia coli</i> W3110/pD<sub>λ</sub>-ZE<sub>DIII</sub> as background. After 20&#xa0;days (20 cycles of dilution), the λ<sub>evo</sub> phage developed a ~ 22% genome deletion affecting the non-essential λ <i>b</i> region, rendering a more stable phage that exhibited fusion proteins D<sub>λ</sub>-ZE<sub>DIII</sub> or D<sub>λ</sub>-GFP but not D<sub>λ</sub>-TD. Despite the λ<sub>evo</sub> system was able to decorate itself with the D<sub>λ</sub>-ZE<sub>DIII</sub> protein, the production of viral particles was ~ 1000-fold lower than the λ wild-type, due to the unexpected D<sub>λ</sub>-ZE<sub>DIII</sub> protein aggregation into bacterial inclusion bodies. Decorated phages (10<sup>6</sup> PFU (plaque forming units)/100&#xa0;µl) were inoculated into BALB/c mice, and subsequent dot blot and Western blot immunoassays proved the production of murine antibodies against ZIKV (Zika virus). This multipurpose λ<sub>evo</sub> phage display platform may be used interchangeably with other more soluble peptides, providing better yields.</p> Key points <p>• <i>λ</i><sub><i>evo</i></sub><i>&#xa0;platform for displaying recombinant peptides.</i></p> <p>• <i>Directed evolution to generate&#xa0;λ</i><sub><i>evo</i></sub><i>&#xa0;with more efficient decoration.</i></p> <p>• <i>Antigenic reaction in BALB/c mice by inoculating&#xa0;λ</i><sub><i>evo</i></sub><i>&#xa0;with recombinant peptides.</i></p>

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A Lambda-evo (λevo) phage platform for Zika virus EDIII protein display

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年1月16日

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  • Honorio Negrete-Méndez,
  • Guadalupe Valencia-Toxqui,
  • Eva Martínez-Peñafiel,
  • Oscar Medina-Contreras,
  • Fernando Fernández-Ramírez,
  • Edgar Morales-Ríos,
  • Luis Janiel Navarro-González,
  • Jesús M. Torres-Flores,
  • Luis Kameyama

摘要

Abstract

One of the most significant bacteriophage technologies is phage display, in which heterologous peptides are exhibited on the virion surface. This work describes the display of λ decorative protein Dλ linked to the E protein domain III of Zika virus (Dλ-ZEDIII), to the GFP protein (Dλ-GFP), or to different domain III epitopes of the EZIKV protein (Dλ-TD), exhibited on the surface of an in vitro evolved lambda phage (λevo). This phage harbors a gene D deletion and was subjected to directed evolution using Escherichia coli W3110/pDλ-ZEDIII as background. After 20 days (20 cycles of dilution), the λevo phage developed a ~ 22% genome deletion affecting the non-essential λ b region, rendering a more stable phage that exhibited fusion proteins Dλ-ZEDIII or Dλ-GFP but not Dλ-TD. Despite the λevo system was able to decorate itself with the Dλ-ZEDIII protein, the production of viral particles was ~ 1000-fold lower than the λ wild-type, due to the unexpected Dλ-ZEDIII protein aggregation into bacterial inclusion bodies. Decorated phages (106 PFU (plaque forming units)/100 µl) were inoculated into BALB/c mice, and subsequent dot blot and Western blot immunoassays proved the production of murine antibodies against ZIKV (Zika virus). This multipurpose λevo phage display platform may be used interchangeably with other more soluble peptides, providing better yields.

Key points

• λevo platform for displaying recombinant peptides.

• Directed evolution to generate λevo with more efficient decoration.

• Antigenic reaction in BALB/c mice by inoculating λevo with recombinant peptides.