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  Optimized production strategy of the major capsid protein HPV 16L1 non-assembly variant in E. coli

Roos, N., Breiner, B., Preuss, L., Lilie, H., Hipp, K., Herrmann, H., et al. (2020). Optimized production strategy of the major capsid protein HPV 16L1 non-assembly variant in E. coli. Protein Expression and Purification, 175: 105690. doi:10.1016/j.pep.2020.105690.

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 Creators:
Roos, N, Author
Breiner, B, Author
Preuss, L, Author
Lilie, H, Author
Hipp, K1, Author           
Herrmann, H, Author
Horn, T, Author
Biener, R, Author
Iftner, T, Author
Simon, C, Author
Affiliations:
1Electron Microscopy, Max Planck Institute for Developmental Biology, Max Planck Society, ou_3375794              

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 Abstract: The capsid of human papillomavirus (HPV) consists of two capsid proteins - the major capsid protein L1 and the minor capsid protein L2. Assembled virus-like particles, which only consist of L1 proteins, are successfully applied as prophylactic vaccines against HPV infections. The capsid subunits are L1-pentamers, which are also reported to protect efficiently against HPV infections in animals. The recombinant production of L1 has been previously shown in E. coli, yeast, insect cells, plants and mammalian cell culture. Principally, in E. coli-based expression system L1 shows high expression yields but the protein is largely insoluble. In order to overcome this problem reported strategies address fusion proteins and overexpression of bacterial chaperones. However, an insufficient cleavage of the fusion proteins and removal of co-purified chaperones can hamper subsequent down streaming. We report a significant improvement in the production of soluble L1-pentamers by combining (I) a fusion of a N-terminal SUMO-tag to L1, (II) the heterologous co-expression of the chaperon system GroEL/ES and (III) low expression temperature. The fusion construct was purified in a 2-step protein purification including efficient removal of GroEL/ES and complete removal of the N-terminal SUMO-tag. The expression strategy was transferred to process-controlled high-cell-density fermentation with defined media according to the guidelines of good manufacturing practice. The produced L1 protein is highly pure (>95%), free of DNA (260:280 = 0.5) and pentameric. The production strategy yielded 5.73 mg of purified L1-pentamers per gram dry biomass. The optimized strategy is a suitable alternative for high yield L1-pentamer production and purification as a cheaper process for vaccine production.

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 Dates: 2020-11
 Publication Status: Published in print
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 Rev. Type: -
 Identifiers: DOI: 10.1016/j.pep.2020.105690
PMID: 32681956
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Title: Protein Expression and Purification
Source Genre: Journal
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Publ. Info: San Diego : Academic Press
Pages: - Volume / Issue: 175 Sequence Number: 105690 Start / End Page: - Identifier: ISSN: 1046-5928
CoNE: https://pure.mpg.de/cone/journals/resource/954922650158