Expression, purification, and characterization of the recombinant exo-1,3-β-glucanase (Exo1) of the pathogenic oomycete Pythium insidiosum

Abstract

Pythiosis is a deadly infectious disease of humans and animals living in tropical and subtropical countries. The causative agent is the oomycete Pythium insidiosum. Treatment of pythiosis is challenging. The use of antimicrobial agents usually fails in the treatment of pythiosis. Many patients undergo surgical removal of an infected organ (i.e., eye, arm, and leg). The immunotherapeutic vaccine, prepared from the crude extract of P. insidiosum, shows limited efficacy against pythiosis. The fatal outcome occurs in patients with advanced disease. There are urgent needs for an effective therapeutic modality for pythiosis. Recently, the exo-1,3-β-glucanase (Exo1) has been identified as a conserve immunoreactive protein of P. insidiosum. Exo1 was predicted to reside at the cell membrane and hydrolyze cell wall β-glucan during cell growth. An Exo1 ortholog is absent in the human genome, making it an appealing target for drug or vaccine development. We attempted to clone and express the codonoptimized exo1 gene of P. insidiosum in E. coli. To solve the inclusion body formation, expression and purification of Exo1 were achievable in the denaturing condition using SDS-and urea-based buffers. Exo1 lacked hydrolytic activity due to the absence of proper protein folding and post-translational modifications. ELISA and Western blot analyses demonstrated the immunoreactivity of Exo1 against pythiosis sera. In conclusion, we successfully expressed and purified the immunoreactive Exo1 protein of P. insidiosum. The recombinant Exo1 can be produced at an unlimited amount and could serve as an extra protein to enhance the effectiveness of the current form of the vaccine against pythiosis.

From Cancer Therapy to Winemaking: The Molecular Structure and Applications of β-Glucans and β-1, 3-Glucanases

“…β-1,3-glucanases are proteins with a typical molecular mass of around 30 to 50 kDa, but their sizes lay in a wide range of 12 kDa (e.g., Citrus aurantiifolia ) up to 230 kDa (e.g., Aspergillus nidulans ), with the largest members mostly belonging to eukaryotic species [ 34 , 71 , 74 , 75 , 76 , 77 ]. Typical optimal pH values for β-1,3-glucanase activity are on the slightly acidic range of 5.0 to 6.5, but can range anywhere between 4.0 (e.g., fungi Lentinula edodes [ 78 ]) and 9.0 (e.g., armyworm Spodoptera frugiperda [ 79 ]).…”

“…Fungal endo-β-1,3-glucanases, on the other hand, are more widely distributed across families but mostly belong to GH81. Classified endo-β-1,3(4)-glucanases (EC 3.2.1.6) are either produced by bacteria or fungi and most have been assigned to GH family 16 ( Table 1 ) [ 71 , 76 , 84 , 85 , 86 , 87 ].…”