Optimizing Primary Isolation of Recombinant HPV L1 Protein type 58 from Hansenula polymorpha

Abstract

Human Papillomavirus (HPV) infection is a primary cause of cervical cancer. While HPV types 16 and 18 are globally recognized, HPV 58 is the predominant genotype contributing to cervical cancer incidence in Thailand. Effective prevention is offered by virus-like particle (VLP) vaccines from recombinant L1 capsid protein. However, the high cost of imported vaccines limits their affordability in Thailand, highlighting the need for domestic production to enhance accessibility and self-sustainability. Despite efficient HPV L1 protein production in large-scale bioreactors, a scalable and robust purification process remains challenging, particularly when the protein is expressed in Hansenula polymorpha. This study focused on optimizing the initial isolation of recombinant HPV58 L1 protein from Hansenula polymorpha. Cell disruption was optimized using a high-pressure homogenizer at 1000 bars. The highest L1 protein yield (76.16 µg/mL) was achieved after five homogenization passes. Agitation of the homogenate for 6 h further enhanced the L1 protein yield to 165.67 µg/mL by solubilizing protein trapped within organelles or cell debris. Protein precipitation for L1 isolation was compared using ammonium sulfate (40-50% saturation), PEG6000 (10-25% v/v), and PEG8000 (10-25% v/v). Precipitation with 45% saturated ammonium sulfate at 4°C yielded the highest specific L1 protein (45.9 µg/mL) with fewer impurities. The L1 protein recovery rate ranged from 44% to 57%. This primary isolation provides a crude L1 protein extract for further downstream purification using methods such as chromatography. The purified L1 protein can self-assemble into VLPs and can be formulated into an HPV vaccine. This work supports scalable downstream processes for industrial HPV vaccine production.