Comparative characterization of constitutive and inducible expression systems for human papillomavirus type 18 L1 protein in Kluyveromyces lactis

Background. The use of prophylactic HPV vaccines has demonstrated effectiveness in reducing the incidence of cervical cancer. The yeast Kluyveromyces lactis is a promising platform to produce recombinant vaccine antigens, owing to its GRAS status, eukaryotic post translational modification capabilities, and the low cost nutrient media.

Aim. To develop a producer strain based on Kluyveromyces lactis for the production of recombinant human papillomavirus type 18 L1 protein.

Materials and Methods. For the expression of the HPV18 L1 gene, two integrative vectors were constructed. The first, pKB-HPV18, with a constitutive GAP promoter, and the second, pKB-HPV18, with an inducible LAC4 promoter. K. lactis cells were transformed with the obtained vectors by electroporation. Recombinant strains were cultivated in YEPm medium. Protein synthesis induction for the K. lactis/pKB-HPV18 strain was performed using lactose. The target protein was isolated from cell lysates by ion exchange chromatography (SP Seplife). Protein identification was carried out using liquid chromatography–tandem mass spectrometry (LC MS/MS). All experiments were performed in three biological replicates. Statistical significance was assessed using Student’s t-test (p < 0.05).

Results. Producer strains were obtained for both constructs. The biomass yield was 20 g/L for K. lactis/pKB-HPV18 and 34 g/L for K. lactis/pKB-HPV18. The purity of the target protein after a single ion-exchange chromatography step reached 70–80% for the constitutive system and approached 90% for the inducible system. The identity and structural integrity of the obtained protein were confirmed using LC MS/MS.

Conclusion. Two K. lactis producer strains capable of synthesizing HPV18 L1 protein were successfully constructed. The system with the inducible LAC4 promoter showed higher biomass yield and target protein purity compared to the constitutive system. These results provide a foundation for the further development of an affordable production system for the HPV18 vaccine antigen.

1. Liu F, Xu J, Yang R, Liu S, Hu S, Yan M, et al. New light on treatment of cervical cancer: Chinese medicine monomers can be effective for cervical cancer by inhibiting the PI3K/Akt signaling pathway. Biomedicine and Pharmacotherapy. 2023; 157: 114084. doi: 10.1016/j.biopha.2022.114084

2. Wu J, Jin Q, Zhang Y, Ji Y, Li J, Liu X, et al. Global burden of cervical cancer: current estimates, temporal trend and future projections based on the GLOBOCAN 2022. Journal of the National Cancer Center. 2025; 5(3): 322–329. doi: 10.1016/j.jncc.2024.11.006

3. Afanasiev MS, Dushkin AD, Afanasiev SS, Nesvizhsky YV, Grishacheva TG, Biryukova ES, et al. Type profile of high oncogenic risk human papillomavirus in clinical forms of cervical infection in Russian Federation. Russian Journal of Infection and Immunity = Infektsiya i immunitet. 2025: 15(2): 271–286. (In Russ.). doi: 10.15789/2220-7619-tpo17690

4. Cascardi E, Cazzato G, Daniele A, Silvestris E, Cormio G, Di Vagno G, et al. Association between cervical microbiota and HPV: could this be the key to complete cervical cancer eradication? Biology. 2022;11(8): 1114. doi: 10.3390/biology11081114

5. Zhou D, Xue J, Sun Y, Zhu L, Zhao M, Cui M, et al. Patterns of single and multiple HPV infections in female: A systematic review and meta-analysis. Heliyon. 2024; 10(17): e35736. doi: 10.1016/j.heliyon.2024.e35736

6. Wang R, Huang H, Yu C, Li X, Wang Y, Xie L. Current status and future directions for the development of human papillomavirus vaccines. Frontiers in Immunology. 2024; 15: 1362770. doi: 10.3389/fimmu.2024.1362770

7. Palatty PL, Sacheendran D, Jayachandran M. Human Papilloma Virus Vaccines. In: Baloh Z (ed.). Confronting HPV: Insights and Solutions. Books on Demand; 2025: 77–78. doi: 10.5772/intechopen.1004419

8. Brotherton JML, LaMontagne DS, Bloem PJN. Global status of HPV vaccination two decades in: effective, safe and preventing cancer. Expert Rev Vaccines. 2026; 25(1): 2609869. doi: 10.1080/14760584.2025.2609869

9. Shivakumar T, Das A, Bussoletti Panizo MV, Storch M, Thavarajah W, Freemont PS, et al. A Lactose-Based Kluyveromyces lactis Cell-Free Protein Synthesis System. Chem Bio Eng. 2025; 2(10): 602–611. doi: 10.1021/cbe.5c00011

10. Spohner SC, Schaum V, Quitmann H, Czermak P. Kluyveromyces lactis: an emerging tool in biotechnology. Journal of Biotechnology. 2016; 222: 104–116. doi: 10.1016/j.jbiotec.2016.02.023

11. Ohstrom AM, Buck AE, Du X, Wee J. Evaluation of Kluyveromyces spp. for conversion of lactose in different types of whey from dairy processing waste into ethanol. Frontiers in Microbiology. 2023; 14: 1208284. doi: 10.3389/fmicb.2023.1208284

12. Liao L, Shen X, Li J, Du G, Chen J, Zhang G. A dual strategy of metabolic engineering and multisite integration for enhanced lactase production in Kluyveromyces lactis. Chemical Engineering Journal. 2026; 531: 174271. doi: 10.1016/j.cej.2026.174271

13. Eng JK, Jahan TA, Hoopmann MR. Comet: an open source MS/MS sequence database search tool. Proteomics. 2013; 13(1): 22–24. doi: 10.1002/pmic.201200439

14. Mattanovich D, Branduardi P, Dato L, Gasser B, Sauer M, Porro D. Recombinant protein production in yeasts. Methods in Molecular Biology. 2012; 824: 329–358. doi: 10.1007/978-1-61779-433-9_17

15. Porro D, Gasser B, Fossati T, Maurer M, Branduardi P, Sauer M, et al. Production of recombinant proteins in yeasts: when are these systems better than bacterial production systems? Applied Microbiology and Biotechnology. 2011; 89(4): 939–948. doi: 10.1007/s00253-010-3019-z

16. Chen YM, Liu YuCh, Zhang GP, et al. Human papillomavirus L1 protein expressed in Escherichia coli self-assembles into virus-like particles that are highly immunogenic. Virus research. 2016; 220: 97–103. doi: 10.1016/j.virusres.2016.04.017

17. Rosmeita CN, Budiarti S, Mustopa AZ, et al. Expression, purification, and characterization of self-assembly virus-like particles of capsid protein L1 HPV 52 in Pichia pastoris GS115. Journal of Genetic Engineering and Biotechnology. 2023; 21(1): 126. doi: 10.1186/s43141-023-00571-0

18. Gashti AB, Patel M, Chahal PS, et al. Purification and functional characterization of gag-spike virus-like particles: Process optimization for efficient vaccine production. Vaccine. 2025; 62: 127500. doi: 10.1016/j.vaccine.2025.127500

19. Jensen ON. Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. Current opinion in chemical biology. 2004; 8(1): 33–41. doi: 10.1016/j.cbpa.2003.12.009

20. Sampaio FC, et al. Cheese whey permeate fermentation by Kluyveromyces lactis: a combined approach to wastewater treatment and bioethanol production. Environmental technology. 2020; 41(24): 3210–3218. doi: 10.1080/09593330.2019.1604813