Abstract
Optimizing appropriate signal peptides in mammalian cell-based protein production is crucial given that most recombinant proteins produced in mammalian cells are thought to be secreted proteins. Until now, most studies on signal peptide in mammalian cells have replaced native signal peptides with well-known heterologous signal peptides and bioinformatics-based signal peptides. In the present study, we successfully established an in vitro screening system for synthetic signal peptide in CHO cells by combining a degenerate codon-based oligonucleotides library, a site-specific integration system, and a FACS-based antibody detection assay. Three new signal peptides were screened using this new screening system, confirming to have structural properties as signal peptides by the SignalP web server, a neural network-based algorithm that quantifies the signal peptide-ness of amino acid sequences. The novel signal peptides selected in this study increased Fc-fusion protein production in CHO cells by increasing specific protein productivity, whereas they did not negatively affect cell growth. Particularly, the SP-#149 clone showed the highest qp, 0.73 ± 0.01 pg/cell/day from day 1 to day 4, representing a 1.47-fold increase over the native signal peptide in a serum-free suspension culture mode. In addition, replacing native signal peptide with the novel signal peptides did not significantly affect sialylated N-glycan formation, N-terminal cleavage pattern, and biological function of Fc-fusion protein produced in CHO cells. The overall results indicate the utility of a novel in vitro screening system for synthetic signal peptide for mammalian cell-based protein production.
Key points
• An in vitro screening system for synthetic signal peptide in mammalian cells was established
• This system combined a degenerate codon-based library, site-specific integration, and a FACS-based detection assay
• The novel signal peptides selected in this study could increase Fc-fusion protein production in mammalian cells
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Data availability
The data that support the findings of this study are available on request from the corresponding author. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2022) partner repository with the dataset identifier PXD033441.
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Funding
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2020R1A2C4001982) and the Establishment of Technology Commercialization Collaboration Platform through Korea Innovation Foundation funded by the Ministry of Science and ICT (Project name: Establishing Project on the Collaboration Platform of the Technology-Commercialization for Biomedical-Industry in INNOPOLIS Daedeok/Project number: 2021-DD-RD-0012).
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JHP, GML, and YGK designed the research. GML and YGK supervised the project. JHP, HML, and EJJ conducted the main experiments for signal peptide screening. EJL and YJK contributed to the protein purification and protein quality analyses. SK and SSY contributed to the construction of the expression vector and protein activity assays. JHP, GML, and YGK wrote the manuscript. All authors read and approved the manuscript.
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Park, JH., Lee, HM., Jin, EJ. et al. Development of an in vitro screening system for synthetic signal peptide in mammalian cell-based protein production. Appl Microbiol Biotechnol 106, 3571–3582 (2022). https://doi.org/10.1007/s00253-022-11955-6
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DOI: https://doi.org/10.1007/s00253-022-11955-6