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Microbial Pathways of Lipid A Biosynthesis
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
Paul D. Rick, Christian R. H. Raetz
Lipid A synthesis is initiated by the transfer of the (R)-3-hydroxymyristate moiety from (R)-3-hydroxymyristoyl-acyl-carrier-protein (ACP) to the 3′-hydroxyl of UDP-GlcNAc to yield UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetyl-D-glucosamine (UDP-3-O-monoacyl-N-acetylglucosamine) (48). This reaction is catalyzed by the cytosolic enzyme UDP-N-acetylglucosamine acyltransferase (LpxA) (48,49). The IpxA gene has been cloned and sequenced, and it is located clockwise of dnaE at min 4 on the E. coli chromosome as part of a complex operon that includes three other genes involved in lipid A synthesis (lpxB,fabZ, and firA[lpxD]) as well as several additional open reading frames of unknown function (49–52). The deduced amino acid sequence of LpxA revealed it to be a 28 kDa protein comprised of 262 amino acids (50), and the enzyme has been purified to near homogeneity (53). Indeed, the x-ray crystal structure of the enzyme has been determined to 2.6 Å resolution, and an atomic model of LpxA based on the crystallographic data revealed the enzyme to be comprised of three identical subunits and a new kind of helical protein fold (54). The active site appears to be situated between the subunits. LpxA is the only acyltransferase for which a crystal structure is available.
Optical Imaging Probes
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Reconstitution or complementation (i.e., the reconstitution of functional activity rather than the integrity of protein) of a split-reporter protein (firefly and RLucs) were previously used for constructing more robust protein sensors. These fragmented reporters could be used for imaging nuclear receptor translocation into the nucleus or protein-protein interactions in the cell. Initially this was achieved by using intein-driven approach (46,47). Intein (DnaE) is a catalytic subunit of DNA polymerase III. To utilize intein-driven protein splicing, the full length of the nuclear receptor [e.g., androgen receptor (48)], split RLuc, and split DnaE (a protein splicing element) were used. The obtained reporting construct consisted of (i) cytoplasmic receptor–containing fusion protein with C-terminal fragments of DnaE and luciferase and (ii) nucleus-localized fusion counterpart containing N-terminal halves of DnaE and RLuc. These two fusion proteins exhibit no luciferase activity. Upon ligand (dihydrotestosterone) stimulation, the receptor is translocated into the cellular nucleus (46,48) where protein splicing occurs as a consequence of interaction between the splicing junctions of each DnaE fusion fragment. Later it was shown that alternative approaches could be used for RLuc complementation. Rapamycin was shown to drive interaction between FK506-binding protein (FKBP12) mTOR rapamycin-binding domain (FRB) and FKBP12, which if fused to split-RLuc fragments cause the latter to regain some of its catalytic activity (49,50). A similar strategy based on split EGFP protein was also tested for in vivo imaging of subcutaneously injected cells (50).
Genomic diversity of Helicobacter pylori populations from different regions of the human stomach
Published in Gut Microbes, 2022
Daniel James Wilkinson, Benjamin Dickins, Karen Robinson, Jody Anne Winter
Heat maps were produced (Figure 3; Supp. Fig. 21) to combine the key findings from the population consensus whole-genome alignments (Fig. 1A-B; Suppl. Fig. 1–14 A-B) and read mapping (minor allele calling pipeline) approach (Figure 2; Suppl. Fig. 20) and these highlighted the number of differences detected by each approach. Some genes, for example cagY (HP0527) in patient 265, were identified as having differences between the antrum and corpus regions by genome alignment but also had polymorphic diversity within one or both region(s) detected by the minor allele calling pipeline. Other genes were different between the regions (by consensus genome alignment) but showed no within niche diversity at either region e.g. dnaE (HP1460) in patient 120. This shows how both techniques can be used in tandem to better capture the genetic diversity both within and between populations of H. pylori.
Anti-virulence strategies for Clostridioides difficile infection: advances and roadblocks
Published in Gut Microbes, 2020
David Stewart, Farhan Anwar, Gayatri Vedantam
Multiple novel CABs have been synthesized and, by themselves, exhibit poor/negligible antibacterial activity within the concentration ranges needed to deliver their ASO cargo. However, when these CABs were combined with an ASO targeting the dnaE gene of CD (encoding the alpha subunit of DNA polymerase III), potent bacteridical activity was achieved at <12 µg/mL, with no detectable activity on Escherichia coli, Enterococcus faecalis, Bacteroides fragilis, three key representatives of the human commensal microbiota.37
Intein mediated high throughput screening for bispecific antibodies
Published in mAbs, 2020
Tim Hofmann, Johannes Schmidt, Elke Ciesielski, Stefan Becker, Thomas Rysiok, Mark Schütte, Lars Toleikis, Harald Kolmar, Achim Doerner
Subcloning of Npu DnaE genes for secretory expression in mammalia suspension cells: The genes encoding for Npu DnaEN and Npu DnaEC [46] were ordered as gene synthesis from GeneArt (Thermo Fisher Scientific®) as a codon-optimized version for mammalian expression. Genes were modified with a multiple cloning site (mcs) flanking unique restriction sites for antibody fragment subcloning or exchange performed by standard cloning. Enzymes for standard cloning were purchased from New England Biolabs. The Npu DnaEC gene was subcloned into pTT5 vector backbone by using EcoRI and BamHI restriction sites encoding Fc HCs. Npu DnaEC and Npu DnaEN genes were subcloned into pTT5 vector backbone by using BamHI and NotI restriction sites encoding SEED or Fab HCs. Resulting pTT5-NpuDnaEN constructs contained an N-terminal myc-tag followed by a hexahistidine tag downstream of the CH1 domain of Fab HCs. The resulting pTT5-NpuDnaEC construct contained a C-terminal hexahistidine tag followed by a 4x (G4S) linker upstream of the CH2 domain for Fc- or SEED encoding HCs. VH antibody regions encoding anti-CD40, CEACAM5, CEACAM6, c-MET, and EGFR were subcloned into pTT5-NpuDnaEN backbone vector using ApaI and BamHI restriction sites. To avoid homodimer production of SEEDbodies, point mutations were introduced at position 445HY446and 441RF442 to the SEED-GA HC to prevent binding to protein A by the constant region. The mutations allow selective purification of heterodimers, since the SEED-AG HC is known to avoid forming homodimers. All constructs were verified by Sanger sequencing. Plasmids for transient expression were prepared using GenEluteTM HP Plasmid Maxiprep Kit (Sigma-Aldrich) following manufacturer’s instructions.