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Effect of point mutations in translation initiation context on the expression of recombinant human α1-proteinase inhibitor in transgenic tomato plants

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Abstract

The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human α1-proteinase inhibitor (α1-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant α1-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at −3 position eliminated most of the α1-PI expression, while mutation at −3 alone resulted in about sevenfold reduction. The presence of steady-state levels of α1-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.

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Abbreviations

TIC:

Translation initiation context

ELISA:

Enzyme-linked immunosorbent assay

TSP:

Total soluble protein

α1-PI:

α1-proteinase inhibitor

AMV:

Alfalfa mosaic virus

ER:

Endoplasmic reticulum

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Acknowledgments

We are grateful to Director, NBRI, Lucknow for infrastructural support, encouragement and valuable suggestions. We thankfully acknowledge Council of Scientific and Industrial Research (CSIR), India for providing funds and senior research fellowships to SA and SJ. This work was carried out under the CSIR Network Project CMM0004 and OLP 0031.

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Authors and Affiliations

  1. Plant Transgenic Laboratory, National Botanical Research Institute, P.O. Box 436, Rana Pratap Marg, Lucknow, 226001, India

    Saurabh Agarwal, Shweta Jha, Indraneel Sanyal & D. V. Amla

Authors
  1. Saurabh Agarwal
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  2. Shweta Jha
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  3. Indraneel Sanyal
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  4. D. V. Amla
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Correspondence to D. V. Amla.

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Communicated by P. Kumar.

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Agarwal, S., Jha, S., Sanyal, I. et al. Effect of point mutations in translation initiation context on the expression of recombinant human α1-proteinase inhibitor in transgenic tomato plants. Plant Cell Rep 28, 1791–1798 (2009). https://doi.org/10.1007/s00299-009-0779-y

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  • DOI: https://doi.org/10.1007/s00299-009-0779-y

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