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Ivanov, Maksim G. (2023) Recombinant spidroins from infinite circRNA translation. PhD thesis, University of Nottingham.

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Abstract

Spidroins are a diverse family of peptides and the main components of spider silk. They can be used to produce sustainable, lightweight and durable materials for a large variety of medical and engineering applications. Spiders’ territorial behaviour and cannibalism precludes farming them for silk. Recombinant protein synthesis is the most promising way of producing these peptides. However, many approaches have been unsuccessful in obtaining large titres of recombinant spidroins or ones of sufficient molecular weight. The work described here is focused on expressing high molecular weight spidroins from short circular RNA molecules. Mammalian host cells were transfected with designed circular-RNA-producing plasmid vectors. A backsplicing approach was implemented to successfully circularise RNA in a variety of mammalian cell types. This approach could not express any recombinant spidroins based on a variety of qualitative protein assays. Further experiments investigated the reasons behind this.

Additionally, due to the diversity of spidroins in a large number of spider lineages, there are potentially many spidroin sequences left to be discovered. A bioinformatic pipeline was developed that accepts transcriptome datasets from RNA sequencing and uses tandem repeat detection and profile HMM annotation to identify novel sequences. This pipeline was specifically designed for the identification of repeat domains in expressed sequences. 21 transcriptomes from 17 different species, encompassing a wide selection of basal and derived spider lineages, were investigated using this pipeline. Six previously undescribed spidroin sequences were discovered. This pipeline was additionally tested in the context of the suckerin protein family. These proteins have recently been investigated for their potential properties in medicine and engineering including adhesion in wet environments. The computational pipeline was able to double the number of suckerins known to date. Further phylogenetic analysis was implemented to expand on the knowledge of suckerins. This pipeline enables the identification of transcripts that may have been overlooked by more mainstream analysis methods such as pairwise homology searches. The spidroins and suckerins discovered by this pipeline may contribute to the large repertoire of potentially useful properties characteristic of this diverse peptide family.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Spriggs, Keith Goodacre, Sara
Keywords:	circRNA, spidroin, transcriptomics, spider silk, circular RNA translation, RCT, rolling circle translation, IORF
Subjects:	Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	72209
Depositing User:	Ivanov, Maksim
Date Deposited:	22 Jul 2023 04:40
Last Modified:	22 Jul 2023 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/72209

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