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Multiple gene expression in plants using MIDAS-P, a versatile type II restriction-based modular expression vector.

Elizabeth C Pinneh1, Craig J van Dolleweerd2, Kathrin Göritzer1

  • 1Molecular Immunology Unit, Institute for Infection and Immunity, St. George's University of London, London, UK.

Biotechnology and Bioengineering
|March 3, 2022
PubMed
Summary
This summary is machine-generated.

The MIDAS-P vector enables iterative cloning of multiple transcription units (TUs) for plant gene expression. Coexpressing the P19 suppressor enhances expression of multiple anti-HIV genes in plants.

Keywords:
GriffithsinMIDAS-PVRC01anti-HIV cocktailmodulartandem plant expression vector

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Area of Science:

  • Plant Biotechnology
  • Molecular Biology
  • Virology

Background:

  • Developing effective plant-based strategies for producing therapeutic proteins is crucial for combating viral infections like HIV.
  • Efficient expression of multiple genes within a single plant construct presents significant challenges.

Purpose of the Study:

  • To evaluate the efficacy of the MIDAS-P plant expression vector for cloning and expressing multiple anti-HIV therapeutic genes.
  • To investigate the impact of transcription unit (TU) number and the coexpression of RNA silencing suppressors on gene expression levels.

Main Methods:

  • Utilized the MIDAS-P vector for iterative cloning of up to five transcription units (TUs) encoding anti-HIV proteins (VRC01, Griffithsin, J3-VHH) and DsRed reporter.
  • Assembled constructs with varying combinations of 3, 4, or 5 TUs in Nicotiana benthamiana.
  • Assessed mRNA and protein expression levels, with and without coexpression of the P19 RNA silencing suppressor.

Main Results:

  • mRNA levels decreased with more than two TUs per construct.
  • Coexpression of P19 significantly boosted mRNA and protein expression for all components.
  • Gene expression dropped in constructs with four or more TUs, except for DsRed in specific positions within 5 TU constructs.
  • The position of TUs in 3 TU constructs did not influence expression levels.

Conclusions:

  • The MIDAS-P system is effective for simultaneous expression of multiple proteins in a single plant construct.
  • Coexpression of P19 is essential for overcoming expression limitations associated with multiple TUs.
  • The MIDAS-P system facilitates the development of plant-based expression platforms for complex therapeutic cocktails.