Determination of amino acids misincorporation in recombinant protein by mass spectrometry

Authors

  • MZ Alam Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet
  • L Regioneiri RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, Aveiro
  • MAS Santos RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, Aveiro

DOI:

https://doi.org/10.3329/bjas.v42i1.15760

Keywords:

Amino acids misincorporation, mass spectrometry, recombinant protein, ?- galactosidase

Abstract

The synthesis of protein according to genetic code of a gene determines the basis of life and a stable proteome is necessary for cell homeostatis. However, errors occur naturally during translation of protein from its mRNA, which varies from 10-3 to 10-4 per codon. These errors are more frequent in recombinant protein overexpressed in heterologous hosts and affect protein functionality. The increasing amount of nonfunctional protein is often related to mistranslation of a gene under stress. In the present study, Saccharomyces cerevisiae as a host organism to overexpress E. coli lacZ gene fusion with GST to quantify misincorporation of amino acid in GST-? galactosidase recombinant protein. The yeast was treated with various stressors such as ethanol, chromium (CrO3), and aminoglycoside antibiotic - geneticin (G418) to induce protein aggregation. The misincorporation of amino acids was studied in soluble protein fractions by mass-spectrometry to determine how much misincorporation occur. We found that under experimental stress conditions the misincorporation of amino acids ranges from 5.6 ×10-3 to 8 × 10-3, which represents 60-80 fold higher than reported level.

DOI: http://dx.doi.org/10.3329/bjas.v42i1.15760

Bang. J. Anim. Sci. 2013. 42 (1): 11-19

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Published

2013-07-22

How to Cite

Alam, M., Regioneiri, L., & Santos, M. (2013). Determination of amino acids misincorporation in recombinant protein by mass spectrometry. Bangladesh Journal of Animal Science, 42(1), 11–19. https://doi.org/10.3329/bjas.v42i1.15760

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