Self-assembling protein structures for novel applications in synthetic biology

Self-assembling protein tools are highly desirable in the field of synthetic biology. They enable the creation of new macromolecular structures for novel applications, by providing users with the ability to ‘build’ with proteins post-translationally: both in vivo and in vitro. In this thesis, I investigate and describe applications for a range of self-assembling protein tools. These include the BslA protein, SpyTag/SpyCatcher and SnoopTag/SnoopCatcher peptide/protein pairs, mVirD2 protein, and split inteins. For each of these tools I outline their capabilities, and then demonstrate a new application, either individually or in tandem. BslA is an amphiphilic protein that has the remarkable ability to self-assemble into monolayers on hydrophobic glass surfaces. Using a BslA-SpyTag or BslA-SnoopTag fusion protein, I show that BslA monolayers can be formed and subsequently functionalised via these tags with proteins attached to SpyCatcher or SnoopCatcher respectively. I show proof of principle with the attachment of fluorescent proteins to the glass surface via these specific tags. Fluorescent labelling of the BslA monolayer with these proteins provides a better understanding of the monolayer properties, specifically with the movement of proteins within the monolayer. Further expansion of the toolbox for functionalising BslA surfaces is achieved using the mVirD2 protein, which forms a covalent bond with DNA at a specific recognition site. This enables DNA to bind to the glass surface through a series of specific and covalent interactions: first to mVirD2-SpyCatcher fusion protein, and then to BslA-SpyTag. These tools are utilised to test different applications with biotechnological relevance. The applications proposed and tested in this thesis include: screening for new protein/peptide interactions, nanopillars for anti-glint purposes, and real-time cell-free analyte monitoring. Within the cell-free experiments, I focus on the capabilities of a BslA surface to capture proteins expressed in situ, and test cell-free protein expression from DNA covalently-bound to the surface. Following this, I attach protein biosensors to the BslA surface via SpyTag/SpyCatcher peptide/protein pairs and also use them to monitor fluctuations in phosphate levels during a cell-free reaction as it proceeds. I also help with characterisation of an expanded library of split inteins for protein trans-splicing, by testing the efficiency of splicing and comparing the orthogonality of the library in vitro. Subsequently, I demonstrate the use of split inteins in the development of a protocol for the solid-phase assembly of large, extremely repetitive proteins using split intein trans-splicing. I provide two protocols, that enable the ligation of six protein units, using either five orthogonal split inteins simultaneously, or using just two split inteins sequentially. This enables the creation of repetitive proteins to a length that would be difficult to achieve in vivo. In summary, this thesis presents new applications for self-assembling protein systems and further characterises the tools that underpin them

Self-Assembling Protein Surfaces for In Situ Capture of Cell-Free-Synthesized Proteins

We present a new method for the surface capture of proteins in cell-free protein synthesis (CFPS). We demonstrate the spontaneous self-assembly of the protein BslA into functionalizable surfaces on the surface of a CFPS reaction chamber. We show that proteins can be covalently captured by such surfaces, using “Catcher/Tag” technology. Importantly, proteins of interest can be captured either when synthesised in situ by CFPS above the BslA surfaces, or when added as pure protein. The simplicity and cost efficiency of this method suggest that it will find many applications in cell-free-based methods

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely