Biology subjects

Regan, L.

Publications and source records attributed to Regan, L..

7 recordsLinked to original sources

bioRxiv2025

Cell-free protein synthesis as a method to rapidly screen machine learning-directed protease variants

Machine learning (ML) tools have revolutionised protein structure prediction, engineering and design, but the best ML tool is only as good as the training data it learns from. To obtain high quality structural or functional data, protein purification is typically required, which is both time and resource consuming - especially at the scale required to train ML tools. Here, we showcase cell-free protein synthesis (CFPS) as a straightforward and fast tool for screening and scoring the activity of protein variants for ML workflows. We demonstrate the utility of the system by improving the kinetic qualities of a protease. By rapidly screening just 48 random variants to initially sample the fitness landscape, followed by 32 more targeted variants, we identified several protease variants with improved kinetic properties.

synthetic biology↗

bioRxiv2024

A new highly specific and soluble protease for precise removal of N-terminal purification tags

The affinity purification tags have significantly enhanced the ease of purification of many proteins. After purification, it is necessary to remove the affinity purification tag for many applications. Several specific proteases have been developed for tag removal, but none yet has both, a desirable cleavage specificity and high solubility. Here we describe the design and characterization of a novel protease, which we have named Con1, which exhibits desirable cleavage specificity and high solubility. We present a detailed analysis of the kinetic properties of Con1, along with demonstrating its ability to remove purification tags from different proteins.

biochemistry↗

bioRxiv2024

Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT

Super-resolution microscopy has revolutionized biological imaging, enabling the visualization of structures at the nanometer length scale. Its application in live cells, however, has remained challenging. To address this, we adapted LIVE-PAINT, an approach we established in yeast, for application in live mammalian cells. Using the 101A/101B coiled-coil peptide pair as a peptide-based targeting system, we successfully demonstrate the super-resolution imaging of two distinct proteins in mammalian cells, one localized in the nucleus, and the second in the cytoplasm. This study highlights the versatility of LIVE-PAINT, suggesting its potential for live-cell super-resolution imaging across a range of protein targets in mammalian cells. We name the mammalian cell version of our original method mLIVE-PAINT.

synthetic biology↗

bioRxiv2024

Characterisation and comparison of semen microbiota and sperm function in men with infertility, recurrent miscarriage, or proven fertility

Several studies have associated seminal microbiota abnormalities with male infertility but have yielded differing results owing to their limited sizes or depths of analyses. The semen microbiota during recurrent pregnancy loss (RPL) has not been investigated. Comprehensively assessing the seminal microbiota in men with reproductive disorders could elucidate its potential role in clinical management. We used semen analysis, terminal-deoxynucleotidyl-transferase-mediated-deoxyuridine-triphosphate-nick-end-labelling, Comet DNA fragmentation, luminol ROS chemiluminescence and metataxonomic profiling of semen microbiota by16S rRNA amplicon sequencing in this prospective, cross-section study to investigate composition and bacterial load of seminal bacterial genera and species, semen parameters, reactive oxidative species (ROS), and sperm DNA fragmentation in men with reproductive disorders and proven fathers. 223 men were enrolled included healthy men with proven paternity (n=63), the male partners in a couple encountering RPL (n=46), men with male factor infertility (n=58), and the male partners of couples unexplained infertility (n=56). Rates of high sperm DNA fragmentation, elevated ROS and oligospermia were more prevalent in the study group compared with control. In all groups, semen microbiota clustered into three major genera-dominant groups (1, Streptococcus; 2, Prevotella; 3, Lactobacillus and Gardnerella); no species clusters were identified. Group 2 had the highest microbial richness (P<0.001), alpha-diversity (P<0.001), and bacterial load (P<0.0001). Overall bacterial composition or load has not found to associate with semen analysis, ROS or DNA fragmentation. Whilst, global perturbation of the seminal microbiota is not associated with male reproductive disorders, men with unidentified seminal Flavobacterium are more likely to have abnormal seminal analysis. Future studies may elucidate if Flavobacterium reduction has therapeutic potential.

microbiology↗

bioRxiv2023

Imaging proteins sensitive to direct fusions using transient peptide-peptide interactions

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of protein subcellular location and function. Some proteins, however, show altered localization and/or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to [~]200 nm, which is two orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs. FOR TABLE OF CONTENTS ONLY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/547312v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@ce1d9borg.highwire.dtl.DTLVardef@1f9b5c8org.highwire.dtl.DTLVardef@10d92ccorg.highwire.dtl.DTLVardef@20ad5d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Live-cell super-resolution imaging of actin using LifeAct-14 with a PAINT-based approach

We present direct-LIVE-PAINT, an easy-to-implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct-LIVE-PAINT with an actin-binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct-LIVE-PAINT can be used to image actin structures below the diffraction-limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells.

synthetic biology↗

bioRxiv2020

LIVE-PAINT: Super-Resolution Microscopy Inside Live Cells Using Reversible Peptide-Protein Interactions

We present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extend data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗