Heterologous expression and mutagenesis of recombinant Vespa affinis hyaluronidase protein (rVesA2)

 

Prapenpuksiri Rungsa^1  2, Piyapon Janpan^1  2, Yutthakan Saengkun^1  2, Nisachon Jangpromma¹, Sompong Klaynongsruang¹, Rina Patramanon¹, Nunthawun Uawonggul³, Jureerut Daduang⁴, Sakda Daduang^1  2

 

1 Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand

2 Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand

3 Faculty of Science, Nakhon Phanom University, Nakhon Phanom, 48000, Thailand

4 Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand

 

ABSTRACT

Background:

Crude venom of the banded tiger waspVespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors.

Methods:

The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed.

Results:

V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)₇ composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli 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.

Conclusion:

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.

 

Keywords Vespa affinis; Hyaluronidase; Wasp, Venom; Structure analysis; Modelling; Cloning; Protein expression

 

Received: May 21, 2019.

Accepted: October 18, 2019.

 

Correspondence: sakdad@kku.ac.th

 

Competing interests

The authors declare that they have no competing interests.

 

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.