Mathony, Jan (2023)
Engineering Proteins by Domain Insertion.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00023638
Ph.D. Thesis, Primary publication, Publisher's Version
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Item Type: | Ph.D. Thesis | ||||
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Type of entry: | Primary publication | ||||
Title: | Engineering Proteins by Domain Insertion | ||||
Language: | English | ||||
Referees: | Niopek, Prof. Dr. Dominik ; Süß, Prof. Dr. Beatrix | ||||
Date: | 2023 | ||||
Place of Publication: | Darmstadt | ||||
Collation: | xviii, 167 Seiten | ||||
Date of oral examination: | 23 March 2023 | ||||
DOI: | 10.26083/tuprints-00023638 | ||||
Abstract: | Protein domains are structural and functional subunits of proteins. The recombination of existing domains is a source of evolutionary innovation, as it can result in new protein features and functions. Inspired by nature, protein engineering commonly uses domain recombination in order to create artificial proteins with tailor-made properties. Customized control over protein activity, for instance, can be achieved by harnessing switchable domains and functionally linking them to effector domains. Many natural protein domains exhibit conformational changes in response to exogenous triggers. The insertion of light-switchable receptor domains into an effector protein of choice, for instance, allows the control of effector activity with light. The resulting optogenetic proteins represent powerful tools for the investigation of dynamic cellular processes with high precision in time and space. On top, optogenetic proteins enable manifold biotechnological applications and they are even considered potential candidates for future therapeutics. In this study, we first focused on CRISPR-Cas9 genome editing and applied a domain insertion strategy to genetically encoded inhibitors of the CRISPR nuclease from Neisseria meningitidis (NmeCas9), which due to its small size and high DNA sequence-specificity is of great interest for CRISPR genome editing applications. Fusing stabilizing domains to the NmeCas9 inhibitory protein AcrIIC1 allowed us to boost its inhibitory effect, thereby yielding a potent gene editing off-switch. Furthermore, the insertion of the light-responsive LOV2 domain from Avena sativa into AcrIIC3, the most potent inhibitor of NmeCas9, enabled the optogenetic control of gene editing via light-dependent NmeCas9 inhibition. Further investigation of the engineered inhibitors revealed the potential these proteins could have with respect to safe-guarding of the CRISPR technology by selectively reducing off-target editing. The laborious optimization of the engineered CRISPR inhibitors necessary by the time motivated us to more systematically investigate possibilities and constraints of protein engineering by domain insertion using an unbiased insertion approach. Previously, single protein domains were usually introduced only at a few rationally selected sites into target proteins. Here, we inserted up to five structurally and functionally unrelated domains into several different candidate effector proteins at all possible positions. The resulting libraries of protein hybrids were screened for activity by fluorescence-activated cell sorting (FACS) and subsequent next-generation sequencing (Flow-seq). Training machine learning models on the resulting, comprehensive datasets allowed us to dissect parameters that affect domain insertion tolerance and revealed that sequence conservation statistics are the most powerful predictors for domain insertion success. Finally, extending our experimental Flow-seq pipeline towards the screening of engineered, switchable effector variants yielded two potent optogenetic derivatives of the E. coli transcription factor AraC. These novel hybrids will enable the co-regulation of bacterial gene expression by light and chemicals. Taken together, our study showcases the design of functionally diverse protein switches for the control of gene editing and gene expression in mammalian cells and E. coli, respectively. In addition, the generation of a large domain insertion datasets enabled - for the first time - the unbiased investigation of domain insertion tolerance in several evolutionary unrelated proteins. Our study showcases the manifold opportunities and remaining challenges behind the engineering of proteins with new properties and functionalities by domain recombination. |
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Status: | Publisher's Version | ||||
URN: | urn:nbn:de:tuda-tuprints-236381 | ||||
Classification DDC: | 500 Science and mathematics > 570 Life sciences, biology | ||||
Divisions: | Interdisziplinäre Forschungsprojekte > Centre for Synthetic Biology | ||||
Date Deposited: | 04 Apr 2023 12:56 | ||||
Last Modified: | 06 Apr 2023 06:05 | ||||
URI: | https://tuprints.ulb.tu-darmstadt.de/id/eprint/23638 | ||||
PPN: | 506585727 | ||||
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