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The SARS-CoV-2 membrane (M) protein is an attractive target for therapeutic research since it has a low mutational rate and is involved in viral morphogenesis and assembly. This work aimed to optimize the expression of the M protein in Escherichia coli strain BL21 (DE3) by utilizing the low-copy pET28a+ vector. The study discovered that amplification of the M protein gene by PCR produced non-specific bands when a high amount of template DNA was used. Hence, the amount of template was carefully controlled to perform amplification of the M protein gene in pET28a+-M extracted from the transformants. In order to find the ideal circumstances for optimizing the yield of soluble protein, induction with different Isopropyl Thiogalactoside (IPTG) concentrations and temperatures was carried out. The M protein remained mostly insoluble at 18 °C and 37 °C, so bacterial protein extraction reagent (B-PER) was used to solubilize the protein expressed. Since low-copy plasmid decreased the overall protein production, usage of richer media is suggested to obtain large yields of soluble M protein. The findings highlight the necessity for extensive refinement to enhance protein solubility and enable large-scale production.

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Optimization of SARS-CoV-2 M Protein Expression in E. coli BL21 (DE3) Using pET28a+

  • 会议论文

  • 发表日期：

    2025年

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  • Tasshitra Subramaniam,
  • Siti Aisyah Mualif,
  • Teh Yoong Mond

摘要

The SARS-CoV-2 membrane (M) protein is an attractive target for therapeutic research since it has a low mutational rate and is involved in viral morphogenesis and assembly. This work aimed to optimize the expression of the M protein in Escherichia coli strain BL21 (DE3) by utilizing the low-copy pET28a+ vector. The study discovered that amplification of the M protein gene by PCR produced non-specific bands when a high amount of template DNA was used. Hence, the amount of template was carefully controlled to perform amplification of the M protein gene in pET28a+-M extracted from the transformants. In order to find the ideal circumstances for optimizing the yield of soluble protein, induction with different Isopropyl Thiogalactoside (IPTG) concentrations and temperatures was carried out. The M protein remained mostly insoluble at 18 °C and 37 °C, so bacterial protein extraction reagent (B-PER) was used to solubilize the protein expressed. Since low-copy plasmid decreased the overall protein production, usage of richer media is suggested to obtain large yields of soluble M protein. The findings highlight the necessity for extensive refinement to enhance protein solubility and enable large-scale production.