Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg206-Ser207 of the C-terminal fragment, peptide 54–327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme.
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Research Article|
May 14 2007
Tailoring structure–function properties of L-asparaginase: engineering resistance to trypsin cleavage
Georgia A. Kotzia;
Georgia A. Kotzia
1Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855-Athens, Greece
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Katerina Lappa;
Katerina Lappa
1Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855-Athens, Greece
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Nikolaos E. Labrou
Nikolaos E. Labrou
1
1Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855-Athens, Greece
1To whom correspondence should be addressed (email Lambrou@aua.gr).
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Publisher: Portland Press Ltd
Received:
November 15 2006
Revision Received:
February 19 2007
Accepted:
February 21 2007
Accepted Manuscript online:
February 21 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2007 Biochemical Society
2007
Biochem J (2007) 404 (2): 337–343.
Article history
Received:
November 15 2006
Revision Received:
February 19 2007
Accepted:
February 21 2007
Accepted Manuscript online:
February 21 2007
Citation
Georgia A. Kotzia, Katerina Lappa, Nikolaos E. Labrou; Tailoring structure–function properties of L-asparaginase: engineering resistance to trypsin cleavage. Biochem J 1 June 2007; 404 (2): 337–343. doi: https://doi.org/10.1042/BJ20061708
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