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Amino acid substitution on β and α of Cyt2Aa2 affects molecular interaction of protoxin

  • Thammachat, Siriya (Institute of Molecular Biosciences, Mahidol University, Salaya Campus) ;
  • Pungtanom, Nuanwan (Institute of Molecular Biosciences, Mahidol University, Salaya Campus) ;
  • Kidsanguan, Somruathai (Institute of Molecular Biosciences, Mahidol University, Salaya Campus) ;
  • Pathaichindachote, Wanwarang (National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency) ;
  • Promdonkoy, Boonhiang (National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency) ;
  • Krittanai, Chartchai (Institute of Molecular Biosciences, Mahidol University, Salaya Campus)
  • Received : 2009.11.25
  • Accepted : 2010.03.18
  • Published : 2010.06.30

Abstract

Cyt2Aa2 is a mosquito-larvicidal protein produced as a 29 kDa crystalline protoxin from Bacillus thuringiensis subsp. darmstadiensis. To become an active toxin, proteolytic processing is required to remove amino acids from its N- and C-termini. This study aims to investigate the functional role of amino acid residues on the N-terminal ${\beta}1$ and C-terminal ${\alpha}F$ of Cyt2Aa2 protoxin. Mutant protoxins were constructed, characterized and compared to the wild type Cyt2Aa2. Protein expression data and SDS-PAGE analysis revealed that substitution at leucine-33 (L33) of ${\beta}1$ has a critical effect on dimer formation and structural stability against proteases. In addition, amino acids N230 and I233-F237 around the C-terminus ${\alpha}F$ demonstrated a crucial role in protecting the protoxin from proteolytic digestion. These results suggested that ${\beta}1$ and ${\alpha}F$ on the Nand C-terminal ends of Cyt2Aa2 protoxin play an important role in the molecular interaction and in maintaining the structural stability of the protoxin.

Keywords

References

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