Heterologous expression and mutagenesis of recombinant Vespa affinis hyaluronidase protein (rVesA2)
Crude venom of the banded tiger wasp contains a variety of enzymes including hyaluronidases, commonly known as spreading factors. The cDNA cloning, sequence analysis and structural modelling of venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rV...
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Published in | The journal of venomous animals and toxins including tropical diseases Vol. 25; p. e20190030 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English Portuguese |
Published |
Brazil
Centro de Estudos de Venenos e Animais Peçonhentos
2019
Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP/UNESP) SciELO |
Subjects | |
Online Access | Get full text |
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Summary: | Crude venom of the banded tiger wasp
contains a variety of enzymes including hyaluronidases, commonly known as spreading factors.
The cDNA cloning, sequence analysis and structural modelling of
venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed.
venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted
-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)
composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in
with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4.
The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity. |
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Bibliography: | Authors’ contributions: PR and PJ conducted most of the experiments, coordinated the data analysis and drafted the manuscript. YS carried out partial molecular biology techniques. NJ performed the bioinformatics analysis. SK, RP and NU contributed to the study design and editing manuscript. JD performed the molecular analyses and contributed to the editing of the manuscript. SD designed the research and the experiments, coordinated the study, wrote and edited the manuscript. All authors read and approved the final manuscript. Competing interests: The authors declare that they have no competing interests. |
ISSN: | 1678-9199 1678-9199 |
DOI: | 10.1590/1678-9199-JVATITD-2019-0030 |