Cloning and expression of Human Papilloma virus type 16 L1 capsid protein in bacteria

  • Silvia Tri Widyaningtyas
  • Sofy Meilany
  • Budiman Bela
Keywords: L1, HPV 16, cervical cancer


Latar belakang: Secara alamiah protein kapsid L1 Human Papillomavirus (HPV) tipe 16 dapat mengalami auto assembly untuk membentuk Viral like particle (VLP). Terkait dengan penelitian vaksin HPV, VLP dapat digunakan untuk berbagai keperluan seperti vaksin, pseudovirion atau SpyTag-Spycatcher. Penelitian ini ditujukan untuk mendapatkan plasmid rekombinan yang digunakan untuk produksi protein L1 HPV 16.

Metode: Gen penyandi protein L1 HPV 16 diklona ke dalam vector pQE80L, suatu plasmid yang mengandung sistem ekspresi untuk prokariota. DNA penyandi HPV 16 L1 disisipkan pada situs restriksi BamHI dan Hind III plasmid pQE80L. Plasmid rekombinan yang mengandung gen L1 HPV 16dikonfirmasi menggunakan PCR dan analisis enzim restriksi. Lebih lanjut untuk memastikan bahwa gen rekombinan L1 HPV 16 dapat diekspresikan dalam prokariota, plasmid rekombinan ditransformasikan ke bakteri Escherichia coli BL21 (DE3). Bakteri diinduksi dengan Isopropyl β-D-1-thiogalactopyranoside (IPTG) dengan berbagai konsentrasi dan berbagai waktu inkubasi.

Hasil: protein rekombinan L1, berat 56 kDa, telah berhasil diekspresikan dalam sistem prokariota. Protein rekombinan L1 dapat dimurnikan menggunakan TalonR dalam kondisi denaturasi.

Kesimpulan: gen L1 HPV 16 telah dikloning ke dalam pQE80L dan berhasil diekspresikan dalam sistem prokariota. (Health Science Journal of Indonesia 2019;10(2):82-9)

Kata kunci: L1, HPV 16, cervical cancer



Background: Naturally Human Papillomavirus (HPV) type 16 L1 capsid protein can auto assemble to form Viral like particles (VLP). Concerning to vaccine development for HPV, VLP can be used for a variety of needs such as a vaccine, pseudovirion or SpyTag-Spycatcher. In this study, to obtain a vector expression that can be used in the production of HPV L1 protein, we cloned gene coding HPV 16 L1 protein into pQE80L a plasmid contains an expression system for prokaryote.

Methods: The DNA coding HPV 16 L1 was inserted at BamHI and Hind III restriction sites of pQE80L plasmid. The recombinant plasmid containing the HPV L1 gene was confirmed using PCR colony and enzyme restriction. Further to ensure the recombinant HPV 16 L1 gene could be expressed in a prokaryote, the recombinant plasmid was transformed into bacteria Escherichia coli BL21 (DE3). The bacteria were induced with IPTG with various concentrations and various incubation time.

Result: L1 recombinant protein, 56 kDa in weight, has successfully been expressed in prokaryote system. L1 recombinant protein can be purified using TalonR under denaturing conditions.

Conclusion: L1 HPV 16 gene has been cloned into pQE80L and successfully expressed in prokaryote system. (Health Science Journal of Indonesia 2019;10(2):82-9)

Keywords: L1, HPV 16, cervical cancer


Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, et al. Global cancer observatory: cancer today. Lyon, France: International Agency for Research on Cancer. 2018 Available from:

Doorbar J, Egawa N, Griffin H, Kranjec C, Murakami I. Human papillomavirus molecular biology and disease association. Review in Medical. Virology. 2016; 25: 2–23.

Wei M, Wang D, Li Z, Song S, Kong X, Mo X, et al. N-terminal truncations on L1 proteins of human papillomaviruses promote their soluble expression in Escherichia coli and self-assembly in vitro. Emerg Microbes Infect. 2018 Sep 26;7(1):160. doi: 10.1038/ s41426-018-0158-2. PMID: 30254257; PMCID: PMC6156512. 9-6

Bruni L, Barrionuevo-Rosas L, Albero G, Aldea M, Serrano B, Valencia S, et al. Barcelona: ICO information centre on HPV and cancer; Human papillomavirus and related diseases in Senegal. Summary report 2016-02-26. Available from:

WHO/BS/2018.2348. Recommendations to assure the quality, safety and efficacy of recombinant human papillomavirus virus-like particle vaccines. Geneva, 12 to 16 October 2015. Available from: Recommendations_HEP_E_vaccines.pdf?ua=1

Pinidis P, Tsikouras P, Iatrakis G, Zervoudis S, Koukouli Z, Bothou A, et al. Human Papilloma virus life cycle and carcinogenesis. Maedica (Buchar). 2016 Mar;11(1):48-54. PMID: 28465751; PMCID: PMC5394500.

Buck CB, Day PM, Trus BL. The papillomavirus major capsid protein L1. Virology. 2013;445(1-2),169–174. doi:10.1016/j.virol.2013.05.038

Wu X., Ma X., Li Y, Xu Y, Zheng N, Xu S, et al. Induction of neutralizing antibodies by human papillomavirus vaccine generated in mammalian cells. Antibody Therapeutics. 2019;2(2). Pages 45–53,

Bousarghin L. Positively charged sequences of human papillomavirus type 16 capsid proteins are sufficient to mediate gene transfer into target cells via the heparan sulfate receptor. Journal of General Virology. 2003;84(1)157–64. doi:10.1099/vir.0.18789-0

Wu WH, Gersch E, Kwak K, Jagu S, Karanam B, et al. Capsomer vaccines protect mice from vaginal challenge with Human Papillomavirus. PLoSONE 2011 6(1 ): e27141. doi:10.1371/journal.pone.0027141

Bang HB, Lee TH, Lee YJ, Jeong KJ. High-level production of Human Papillomavirus (HPV) type 16 L1 in Escherichia coli. J. Microbiol. Biotechnol. 2016;26(2), 356–363.

Barzegar H, Sharifi H, Langroudi L, Azadmanesh K, Arashkia A. Human papillomavirus genotype 16 pseudovirus production and purification in HEK-293FT cells. Vaccine Research. 2017;4(3 and 4):46-50.

Brune, KD. Plug-and-Display: decoration of virus-like particles via isopeptide bonds for modular immunization. Sci. Rep. 6, 19234; doi: 10.1038/srep19234 (2016).

Diaz D, Care A, Sunna A. Bioengineering strategies for protein-based nanoparticles. Genes (Basel). 2018 Jul 23;9(7):370. doi: 10.3390/genes9070370. PMID: 30041491; PMCID: PMC6071185

LLi L, Fierer JO, Rapoport TA, Howarth M. Structural analysis and optimization of the covalent association between SpyCatcher and a Peptide Tag. Journal of Molecular Biolog. 2014;426(2)309–17. doi:10.1016/j.jmb.2013.10.021.

Ausubel M, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al. Current protocols in molecular biology volumes 1 and 2. John Wiley & Sons, Inc., Media, PA, 1988. mrd.1080010210.

Lu ZJ, Gloor JW, Mathews DH. Improved RNA secondary structure prediction by maximizing expected pair accuracy. RNA. 2009 Oct;15(10):1805-13. doi: 10.1261/rna.1643609. Epub 2009 Aug 24.PMID: 19703939; PMCID:PMC2743040.

Lai D, Proctor JR, Meyer IM. On the importance of cotranscriptional RNA structure formation RNA 2013 19: 1461-73.

Mathews DH. How to benchmark RNA secondary structure prediction accuracy. Methods. 2019 Jun 1;162 163:60-67. doi: 10.1016/j.ymeth.2019.04.003.

Sterk M, Romilly C, Wagner EGH. Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites. Nucleic Acids Res. 2018 May 4; 46(8): 4188–4199. doi: 10.1093/nar/gky073.

Wagner EJ, Burch BD, Godfrey AC, Salzler HR, Duronio R.J, Marzluff WF. A genome-wide RNA interference screen reveals that variant histones are necessary for replication-dependent histone pre-mRNA processing. Mol Cell. 2007 Nov 30;28(4):692-9.

Leamy KA, Assmann SM, Mathews DH, Bevilacqua PC. Bridging the gap between in vitro and in vivo RNA folding. Q Rev Biophys. 2016 Jan;49:e10. doi: 10.1017/S003358351600007X. Epub 2016 Jun 24. PMID: 27658939; PMCID: PMC5269127.

Lai WB, Middelberg. The production of human papillomavirus type 16 L1 vaccine product from Escherichia coli inclusion bodies. Bioprocess and Biosystems Engineering. 2002;25(2)121–8. doi:10.1007/s00449-002.24. Chen Y, Liu Y, Zhang G, Wang A, Dong Z, Qi Y, et al. Human papillomavirus L1 protein expressed in Escherichia coli self assembles into virus-like particles that are highly immunogenic. Virus Res. 2016 Jul 15;220:97-103. doi:10.1016/j.virusres.2016.04.017.

Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact. 2015 Mar 25;14:41. doi: 10.1186/ s12934-015-0222-8. PMID: 25889252; PMCID:PMC4379949.

Singh A, Upadhyay V, Panda AK. Solubilization and refolding of inclusion body proteins. Insoluble Proteins, Microb Cell Fact. 2015 Mar 25;14:41. doi:10.1186/s12934-015-0222-8.27 Hoffmann D, Ebrahimi M, Gerlach D,Salzig D,Czermak P. Reassessment of inclusion body-basedproduction as a versatile opportunity for difficult-toexpress recombinant proteins. Critical Reviews in Biotechnology. 2017;38(5)729–44. doi:10.1080/07388551.2017.1398134.

Zhang W, Carmichael J, Ferguson J, Inglis S, Ashrafian H, Stanley M. Expression of Human Papillomavirus type 16 L1 protein in Escherichia coli: Denaturation, Renaturation, and Selfassembly of virus-like particlesin vitro. Virology 1998;243(2)423–31. doi:10.1006/viro.1998.9050.

Coimbra, E. C., Gomes, F. B., Campos, J. F., D’arc, M., Carvalho, J. C., Mariz, F. C., Freitas, A. C. Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector. Brazilian Journal of Medical and Biological Research 2011 44(12), 1209–1214. doi:10.1590/s0100-879x2011007500141.

Combita AL, Touzé A, Bousarghin L, Christensen ND, Coursaget P. Identification of two crossneutralizing linear epitopes within the L1 major capsid protein of human papillomaviruses. J Virol. 2002;76(13):6480–6. doi:10.1128/jvi.76.13.6480-6486.2002.

Seo PS, Heo SY, Han EJ, Seo JW, Ghim SJ, Kim CH. Bacterial expression and purification of human papillomavirus type 18 L1. Biotechnol. Bioprocess Eng. 2009;14:168-74.

Huang X, Wang X, Zhang J, Xia N, Zhao Q. Escherichia coli-derived virus-like particles in vaccine development. Npj Vaccines 2017 2(1). doi:10.1038/s41541-017-0006-8.

Akuzum B, Kim S, Nguyen TT, Hong J, Lee S, Kim E, et al. L1 recombinant proteins of HPV tested for antibody forming using sera of HPV quadrivalent vaccine. Immune Netw. 2018 Jun;18(3):e19. https://

Wheatley AK, Kent SJ. Prospects for antibodybased universal influenza vaccines in the context of widespread pre-existing immunity. Expert Review of Vaccines. ;14(9):1227–39. doi:10.1586/14760584.2015.1068125.

How to Cite
Widyaningtyas, S., Meilany, S., & Bela, B. (2019). Cloning and expression of Human Papilloma virus type 16 L1 capsid protein in bacteria. Health Science Journal of Indonesia, 10(2), 82-89.