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Open Access 01-12-2024 | Cytokines | Research

Production of recombinant HPV11/16 E6/E7-MBP-His₆ fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood

Authors: Mei-nian Xu, Mei-zhen Zhong, Si-ning Feng, Yan-qin Xu, Xiao-ming Peng, Kang Zeng, Xiao-wen Huang

Published in: Virology Journal | Issue 1/2024

Abstract

Human papillomavirus (HPV) infection poses a significant threat to public health worldwide. Targeting the function of HPV E6 and E7 proteins and activating the host immune response against these proteins represent promising therapeutic strategies for combating HPV-related diseases. Consequently, the efficient production of soluble, high-purity E6 and E7 proteins is crucial for function and host immune response studies. In this context, we selected the pMCSG19 protein expression vector for Escherichia coli to produce soluble MBP-His₆ tagged HPV11/16 E6/E7 proteins, achieving relatively high purity and yield. Notably, these proteins exhibited low toxicity to peripheral blood mononuclear cells (PBMCs) and did not compromise their viability. Additionally, the recombinant proteins were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, indicating their potential to elicit immune responses. In conclusion, our study offers a novel approach for the production of HPV11/16 E6/E7 fusion proteins with relatively high purity and yield. The fusing HPV11/16 E6/E7 proteins to MBP-His₆ tag may serve as a valuable method for large-scale protein production in future research endeavors.

Zhou C, Tuong ZK, Frazer IH. Papillomavirus immune evasion strategies target the infected cell and the local immune system. Front Oncol. 2019;9:682. https://doi.org/10.3389/fonc.2019.00682.CrossRefPubMedPubMedCentral

Almeida AM, Queiroz JA, Sousa F, Sousa A. Cervical cancer and HPV Infection: ongoing therapeutic research to counteract the action of E6 and E7 oncoproteins. Drug Discov Today. 2019;24:2044–57. https://doi.org/10.1016/j.drudis.2019.07.011.CrossRefPubMed

Gomes D, et al. In silico approaches: a way to unveil novel therapeutic drugs for cervical cancer management. Pharmaceuticals. 2021;14:741. https://doi.org/10.3390/ph14080741.CrossRefPubMedPubMedCentral

Welters MJ, et al. Induction of tumor-specific CD4 + and CD8 + T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine. Clin Cancer Res. 2008;14:178–87. https://doi.org/10.1158/1078-0432.CCR-07-1880.CrossRefPubMed

Dorta-Estremera S, et al. Mucosal HPV E6/E7 peptide vaccination in combination with immune checkpoint modulation induces regression of HPV(+) oral cancers. Cancer Res. 2018;78:5327–39. https://doi.org/10.1158/0008-5472.CAN-18-0892.CrossRefPubMedPubMedCentral

Bartolo-Aguilar Y, et al. The potential of cold-shock promoters for the expression of recombinant proteins in microbes and mammalian cells. J Genet Eng Biotechnol. 2022;20:173. https://doi.org/10.1186/s43141-022-00455-9.CrossRefPubMedPubMedCentral

Verma RR, et al. E6 protein of human papillomavirus 16 (HPV16) expressed in Escherichia coli sans a stretch of hydrophobic amino acids, enables purification of GST-DeltaE6 in the soluble form and retains the binding ability to p53. Protein Expr Purif. 2013;92:41–7. https://doi.org/10.1016/j.pep.2013.08.010.CrossRefPubMed

Zanier K, et al. Formation of well-defined soluble aggregates upon fusion to MBP is a generic property of E6 proteins from various human papillomavirus species. Protein Expr Purif. 2007;51:59–70. https://doi.org/10.1016/j.pep.2006.07.029.CrossRefPubMed

Nomine Y, et al. A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein. Protein Eng. 2001;14:297–305. https://doi.org/10.1093/protein/14.4.297.CrossRefPubMed

10.

de Oliveira LM, Morale MG, Chaves AA, Demasi M, Ho PL. Expression, Polyubiquitination, and therapeutic potential of recombinant E6E7 from HPV16 antigens fused to Ubiquitin. Mol Biotechnol. 2017;59:46–56. https://doi.org/10.1007/s12033-016-9990-6.CrossRefPubMed

11.

Granadillo M, et al. Expression, purification and characterization of a recombinant fusion protein based on the human papillomavirus-16 E7 antigen. Springerplus. 2013;2:1–8. https://doi.org/10.1186/2193-1801-2-12.CrossRef

12.

Petrone L, et al. Recombinant HPV16 E7 assembled into particles induces an immune response and specific tumour protection administered without adjuvant in an animal model. J Transl Med. 2011;9:69. https://doi.org/10.1186/1479-5876-9-69.CrossRefPubMedPubMedCentral

13.

Alonso LG, et al. The HPV16 E7 viral oncoprotein self-assembles into defined spherical oligomers. Biochemistry. 2004;43:3310–7. https://doi.org/10.1021/bi036037o.CrossRefPubMed

14.

Raran-Kurussi S, Keefe K, Waugh DS. Positional effects of fusion partners on the yield and solubility of MBP fusion proteins. Protein Expr Purif. 2015;110:159–64. https://doi.org/10.1016/j.pep.2015.03.004.CrossRefPubMedPubMedCentral

15.

Nomine Y, et al. Formation of soluble inclusion bodies by HPV E6 oncoprotein fused to maltose-binding protein. Protein Expr Purif. 2001;23:22–32. https://doi.org/10.1006/prep.2001.1451.CrossRefPubMed

16.

Lenon M, et al. A useful epitope tag derived from maltose binding protein. Protein Sci. 2021;30:1235–46. https://doi.org/10.1002/pro.4088.CrossRefPubMedPubMedCentral

17.

Hu B, et al. Pre-clinical toxicity and immunogenicity evaluation of a MUC1-MBP/BCG anti-tumor vaccine. Int Immunopharmacol. 2016;33:108–18. https://doi.org/10.1016/j.intimp.2016.02.006.CrossRefPubMed

18.

Layman H, et al. Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18. PLoS ONE. 2020;15:e0229672. https://doi.org/10.1371/journal.pone.0229672.CrossRefPubMedPubMedCentral

19.

Raran-Kurussi S, Waugh DS. Expression and purification of recombinant proteins in Escherichia coli with a his(6) or dual his(6)-MBP tag. Methods Mol Biol. 2017;1607:1–15. https://doi.org/10.1007/978-1-4939-7000-1_1.CrossRefPubMedPubMedCentral

20.

Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983;166:557–80. https://doi.org/10.1016/s0022-2836(83)80284-8.CrossRefPubMed

21.

Nguyen MT, et al. Prokaryotic Soluble overexpression and purification of human VEGF165 by fusion to a maltose binding protein tag. PLoS ONE. 2016;11:e0156296. https://doi.org/10.1371/journal.pone.0156296.CrossRefPubMedPubMedCentral

22.

Han R, et al. Influence of human papillomavirus E7 oncoprotein on maturation and function of plasmacytoid dendritic cells in vitro. Virol Sin. 2018;33:493–501. https://doi.org/10.1007/s12250-018-0069-3.CrossRefPubMedPubMedCentral

23.

Bell MR, Engleka MJ, Malik A, Strickler JE. To fuse or not to fuse: what is your purpose? Protein Sci. 2013;22:1466–77. https://doi.org/10.1002/pro.2356.CrossRefPubMedPubMedCentral

24.

Parida PP, Saraswathi D, Mopidevi SMV, Raran-Kurussi S. Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct. Curr Res Struct Biol. 2023;6:100106. https://doi.org/10.1016/j.crstbi.2023.100106.CrossRefPubMedPubMedCentral

25.

Raran-Kurussi S, Sharwanlal SB, Balasubramanian D, Mote KR. A comparison between MBP- and NT* as N-terminal fusion partner for recombinant protein production in E. Coli. Protein Expr Purif. 2022;189:105991. https://doi.org/10.1016/j.pep.2021.105991.CrossRefPubMed

26.

Imai Y, Tsunokawa Y, Sugimura T, Terada M. Purification and DNA-binding properties of human papillomavirus type 16 E6 protein expressed in Escherichia coli. Biochem Biophys Res Commun. 1989;164:1402–10. https://doi.org/10.1016/0006-291x(89)91826-3.CrossRefPubMed

27.

Bonhoure A, et al. One-step affinity purification of fusion proteins with optimal monodispersity and biological activity: application to aggregation-prone HPV E6 proteins. Microb Cell Fact. 2018;17:191. https://doi.org/10.1186/s12934-018-1039-z.CrossRefPubMedPubMedCentral

28.

Bhatwa A, et al. Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications. Front Bioeng Biotechnol. 2021;9:630551. https://doi.org/10.3389/fbioe.2021.630551.CrossRefPubMedPubMedCentral

29.

Kumar A, Sahu U, Kumari P, Dixit A, Khare P. Designing of multi-epitope chimeric vaccine using immunoinformatic platform by targeting oncogenic strain HPV 16 and 18 against cervical cancer. Sci Rep. 2022;12:9521. https://doi.org/10.1038/s41598-022-13442-4.CrossRefPubMedPubMedCentral

30.

Liu S, et al. Expression and immunogenicity of recombinant porcine epidemic diarrhea virus Nsp9. Virology. 2023;587:109861. https://doi.org/10.1016/j.virol.2023.109861.CrossRefPubMed

31.

Jie J, et al. CpG ODN1826 as a promising Mucin1-maltose-binding protein vaccine adjuvant induced DC maturation and enhanced antitumor immunity. Int J Mol Sci. 2018;19:920. https://doi.org/10.3390/ijms19030920.CrossRefPubMedPubMedCentral

32.

Fang F, et al. MUC1 and maltose–binding protein recombinant fusion protein combined with bacillus calmette–guerin induces MUC1–specific and nonspecific anti–tumor immunity in mice. Mol Med Rep. 2014;10:1056–64. https://doi.org/10.3892/mmr.2014.2306.CrossRefPubMed

33.

Ling J, et al. A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity. Mol Immunol. 2019;114:513–23. https://doi.org/10.1016/j.molimm.2019.08.008.CrossRefPubMedPubMedCentral

Title: Production of recombinant HPV11/16 E6/E7-MBP-His6 fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood
Authors: Mei-nian Xu
Mei-zhen Zhong
Si-ning Feng
Yan-qin Xu
Xiao-ming Peng
Kang Zeng
Xiao-wen Huang
Publication date: 01-12-2024
Publisher: BioMed Central
Keywords: Cytokines
Human Papillomavirus
Published in: Virology Journal / Issue 1/2024
Electronic ISSN: 1743-422X
DOI: https://doi.org/10.1186/s12985-023-02281-y

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