Production of Vespa tropica Hyaluronidase by Pichia pastoris

Abstract

Hyaluronidases have been a subject of great interest in medical and cosmeceutical applications. Previously, our group demonstrated that the venom glands of Vespa tropica contain hyaluronidase enzymes (VesT2s), and heterologous expression of the corresponding gene (VesT2a) in E. coli systems results in inclusion bodies, necessitating functional folding using urea. Here, we report the successful heterologous expression of VesT2a in the Pichia pastoris expression system, with gene construction achieved using GoldenPiCS. After confirming gene integration in the yeast genome, methanol-induced cultures yielded an exceptional amount of VesT2a, approximately two-fold higher than that obtained with the constitutive expression vector (PGAP). Upon culturing in a bioreactor, yeast cells harboring pAOX1-αMF-VesT2a produced secreted proteins with a total yield of 96.45 mg/L. The secreted VesT2a has a molecular weight of 59.35 kDa, significantly higher than the expected molecular weight (~40.05 kDa), presumably due to endogenous glycosylation by the yeast cells. It exhibits optimal activity at 37 °C and pH 3, showing a specific activity of 4238.37 U/mg, and remains active across a broad range of pH and temperature. Notably, it exhibits higher hyaluronidase activity than the crude venom and E. coli-expressed protein, likely due to improved folding via endogenous post-translational modifications, such as disulfide bonds and N-glycosylation; this underscores the potential of heterologous systems for producing venomous hyaluronidases from other species. In silico docking-based analyses further support its catalytic activity and provide insights into seeking natural inhibitors from phenolic-rich plant extracts to alleviate symptoms in patients suffering from insect bites and stings.