Wilkinson, P. orcid.org/0000-0001-9360-1486, Jackson, B. orcid.org/0000-0002-4917-1199, Fermor, H. et al. (1 more author) (2024) A new mRNA structure prediction based approach to identifying improved signal peptides for bone morphogenetic protein 2. BMC Biotechnology, 24. 34. ISSN 1472-6750
Abstract
Background
Signal peptide (SP) engineering has proven able to improve production of many proteins yet is a laborious process that still relies on trial and error. mRNA structure around the translational start site is important in translation initiation and has rarely been considered in this context, with recent improvements in in silico mRNA structure potentially rendering it a useful predictive tool for SP selection. Here we attempt to create a method to systematically screen candidate signal peptide sequences in silico based on both their nucleotide and amino acid sequences. Several recently released computational tools were used to predict signal peptide activity (SignalP), localization target (DeepLoc) and predicted mRNA structure (MXFold2). The method was tested with Bone Morphogenetic Protein 2 (BMP2), an osteogenic growth factor used clinically for bone regeneration. It was hoped more effective BMP2 SPs could improve BMP2-based gene therapies and reduce the cost of recombinant BMP2 production.
Results
Amino acid sequence analysis indicated 2,611 SPs from the TGF-β superfamily were predicted to function when attached to BMP2. mRNA structure prediction indicated structures at the translational start site were likely highly variable. The five sequences with the most accessible translational start sites, a codon optimized BMP2 SP variant and the well-established hIL2 SP sequence were taken forward to in vitro testing. The top five candidates showed non-significant improvements in BMP2 secretion in HEK293T cells. All showed reductions in secretion versus the native sequence in C2C12 cells, with several showing large and significant decreases. None of the tested sequences were able to increase alkaline phosphatase activity above background in C2C12s. The codon optimized control sequence and hIL2 SP showed reasonable activity in HEK293T but very poor activity in C2C12.
Conclusions
These results support the use of peptide sequence based in silico tools for basic predictions around signal peptide activity in a synthetic biology context. However, mRNA structure prediction requires improvement before it can produce reliable predictions for this application. The poor activity of the codon optimized BMP2 SP variant in C2C12 emphasizes the importance of codon choice, mRNA structure, and cellular context for SP activity.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Author(s) 2024. This is an open access article under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. |
Keywords: | Signal peptides; Bone morphogenetic protein; RNA structure; Protein engineering; Synthetic biology; Gene therapy; Regenerative medicine |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Mechanical Engineering (Leeds) ?? Leeds.RC-BISV ?? The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Dentistry (Leeds) > Oral Biology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 19 Sep 2024 16:16 |
Last Modified: | 19 Sep 2024 16:16 |
Status: | Published |
Publisher: | BMC |
Identification Number: | 10.1186/s12896-024-00858-1 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:217378 |
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Filename: A new mRNA structure prediction based approach.pdf
Licence: CC-BY 4.0