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Rifampicin Increases Expression of Plant Codon-Optimized Bacillus thuringiensis δ-Endotoxin Genes in Escherichia coli

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Abstract

Transgenic crops expressing Cry δ-endotoxins of Bacillus thuringiensis for insect resistance have been commercialized worldwide with increased crop productivity and spectacular socioeconomic gains. To attain the enhanced level of protein expression, the cry genes have to be extensively modified for RNA stability and translation efficiency in the plant systems. However, such modifications in nucleotide sequences make it difficult to express the cry genes in Escherichia coli because of the presence of E. coli rare codons. Induction of gene expression through the T7 promoter/lac operator system results in high levels of transcription but limits the availability of activated tRNA corresponding to rare codons that leads to translation stalling at ribosomes. In the present study, an Isopropyl ß-D-1-thiogalactopyranoside (IPTG)/rifampicin combination-based approach was adopted to induce transcription of cry genes through T7 promoter/lac operator while simultaneously inhibiting the transcription of host genes through rifampicin. The results show that the IPTG/rifampicin combination leads to high-level expression of four plant codon-optimized cry genes (cry2Aa, cry1F, cry1Ac, and cry1AcF). Northern blot analysis of the cry gene expressing E. coli samples showed that the RNA expression level in the IPTG-induced samples was higher as compared to that in the IPTG/rifampicin-induced samples. Diet overlay insect bioassay of IPTG/rifampicin-induced Cry toxins with Helicoverpa armigera larvae showed bioactivity (measured as LC50) similar to the previous studies. The experiment has proved that recombinant synthetic gene (plant codon-optimized gene) with the combination of Rifampicin which inhibits DNA-dependent bacterial RNA polymerase and reduces the excessive baggage of translational machinery of the bacterial cell triggers the production of synthetic protein. Purification of protein using high pH buffer increases the solubility of the protein. Further, LC50 analysis shows no reduction of protein activity leads to protein stability. Further, purified cry toxin protein can be used for crop protection against pests and a purified form of the synthetic protein can be used for antibody production and perform the immunoassay for the identification of the transgenic plant. The crystallographic structure of synthetic protein could be used for interaction study with another insect to see insecticidal activity.

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Abbreviations

IMAC:

Immobilized metal-affinity chromatography

LB:

Luria–Bertani

Cry:

Crystal

IPTG:

Isopropyl–d-1-thiogalactopyranoside

mAb:

Monoclonal antibody

PVDF:

Polyvinylidene difluoride membrane

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Acknowledgements

The authors thank ICAR-Network Project on Transgenic Crops for financial support and sincerely acknowledge all the institutions involved in this study.

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Authors and Affiliations

  1. ICAR-National Research Centre On Plant Biotechnology, New Delhi, India

    Vivek Kumar Singh, Mullapudi Lakshmi Venkata Phanindra, Sellamuthu Gothandapani, Sushil Satish Chhapekar & Rohini Sreevathsa

  2. School of Biotechnology, Gautam Buddha University, Greater Noida, India

    Vikrant Nain

  3. Department of Biotechnology, Acharya Nagarjuna University, Guntur, India

    K. R. S. Sambasiva Rao

  4. ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad, India

    Polumetla Ananda Kumar

  5. Department of Biotechnology, National Institute of Technology, Raipur, India

    Vivek Kumar Singh & Awanish Kumar

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  1. Vivek Kumar Singh
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Contributions

VKS, VN, MLVP, PAK designed the experiment; VKS performed most of the experiments, VN, MLVP, and SSC were involved in data analysis; SG performed the western blotting experiment; VKS, RS, KRSSR, PAK, and AK were involved in designing, manuscript editing and finalizing.

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Correspondence to Polumetla Ananda Kumar or Awanish Kumar.

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Singh, V.K., Nain, V., Phanindra, M.L.V. et al. Rifampicin Increases Expression of Plant Codon-Optimized Bacillus thuringiensis δ-Endotoxin Genes in Escherichia coli. Protein J 41, 327–336 (2022). https://doi.org/10.1007/s10930-022-10043-y

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