Fami Israyusnita

Indonesia

COMPARISON OF PICHIA PASTORIS AND HANSENULA POLYMORPHA AS L1 PROTEIN HPV45 VIRUS LIKE PARTICLE EXPRESSION PLATFORM

Fami Israyusnita1,2, Astrid Shabrina Kusumareswari3, Astutiati Nurhasanah1*, Nihayatul Karimah1

1. Centre for Vaccine and Drugs Research, National Research and Innovation Agency (BRIN), Banten 15314, Indonesia
2. Master Program of Biomedical Science, Faculty of Medicine, Universitas Indonesia, Daerah Khusus Ibukota Jakarta 10430, Indonesia
3. Department of Biology, Faculty of Mathematics and Science, Universitas Negeri Malang, Malang, 65145, Jawa Timur, Indonesia

Abstract

Background

Human papillomavirus type 45 (HPV45) is associated with cervical cancer, and its L1 protein is a key component in virus-like particle (VLP)-based prophylactic vaccines. Yeast expression systems offer a cost-effective and scalable platform for VLP production. In this study, we compared vector integration levels and L1HPV45 protein expression in recombinant P. pastoris (pGAPZA-L1HPV45) and H. polymorpha (pHIPH4-L1HPV45) transformants.

Methods

The pGAPZA vector, driven by the constitutive GAP promoter, and the pHIPH4 vector, regulated by the inducible MOX promoter, were introduced into their respective host yeasts. Quantitative real-time PCR (qRT-PCR) was used to determine gene copy number, while protein expression levels were analysed by Western Blot densitometry. Post-transformational vector amplification (PTVA) was applied to P. pastoris transformants to enhance gene copy number and expression. Growth profiling was conducted to evaluate the expression stability of the transformants under selective conditions.

Results

Our findings indicate that in comparison to the P. pastoris pGAPZA-L1HPV45, H. polymopha pHIPH4-L1HPV45 reached maximum expression much earlier (27 h), as compared to 72 h. The results also demonstrate that P. pastoris transformants with increased vector copies exhibited enhanced expression levels, whereas H. polymorpha transformants displayed robust inducible expression with methanol supplementation. P. pastoris pGAPZA-L1HPV45 transformants, which both contained only one copy of L1HPV45 gene expressed the protein at similar level. In H. polymorpha pHIPH4-L1-HPV45 that observed slight copy number variations in Clones 3 (1-2 copies), 4 (1 copy), and 5 (1-3 copies), analysis of WB band intensity suggested that Clone 3 expressed the highest amount of protein.

Conclusions

Based on the results, P. pastoris transformants with increased vector copies exhibited enhanced expression levels, whereas H. polymorpha transformants displayed robust inducible expression with methanol supplementation. These results highlight the potential of both yeast systems for HPV45 L1 protein production, offering insights into optimal strategies for recombinant protein expression in vaccine development.