29 results on '"Nesbeth, D."'
Search Results
2. Towards an Aspect-Oriented Design and Modelling Framework for Synthetic Biology
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Boeing, P., Leon, M., Nesbeth, D., Finkelstein, A., and Barnes, C.
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QA75 ,SynBioWeaver ,host context ,lcsh:Chemical technology ,Article ,lcsh:Chemistry ,QH301 ,lcsh:QD1-999 ,lcsh:TP1-1185 ,CAD ,synthetic biology ,mathematical modelling ,aspect-oriented software engineering ,modularity - Abstract
Work on synthetic biology has largely used a component-based metaphor for system construction. While this paradigm has been successful for the construction of numerous systems, the incorporation of contextual design issues—either compositional, host or environmental—will be key to realising more complex applications. Here, we present a design framework that radically steps away from a purely parts-based paradigm by using aspect-oriented software engineering concepts. We believe that the notion of concerns is a powerful and biologically credible way of thinking about system synthesis. By adopting this approach, we can separate core concerns, which represent modular aims of the design, from cross-cutting concerns, which represent system-wide attributes. The explicit handling of cross-cutting concerns allows for contextual information to enter the design process in a modular way. As a proof-of-principle, we implemented the aspect-oriented approach in the Python tool, SynBioWeaver, which enables the combination, or weaving, of core and cross-cutting concerns. The power and flexibility of this framework is demonstrated through a number of examples covering the inclusion of part context, combining circuit designs in a context dependent manner, and the generation of rule, logic and reaction models from synthetic circuit designs.
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- 2018
3. Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication
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Antoniou, A, Lenart, I, Kriston-Vizi, J, Iwawaki, T, Turmaine, M, McHugh, K, Ali, S, Blake, N, Bowness, P, Bajaj-Elliott, M, Gould, K, Nesbeth, D, Powis, S, University of St Andrews. Cellular Medicine Division, University of St Andrews. Biomedical Sciences Research Complex, University of St Andrews. School of Medicine, and University of St Andrews. Centre for Biophotonics
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X-Box Binding Protein 1 ,NDAS ,Arthritis, Reactive ,Cell Line ,1117 Public Health and Health Services ,lipids ,ankylosing spondylitis ,Humans ,infections ,R2C ,HLA-B27 Antigen ,Cell Cycle ,Salmonella enterica ,Epithelial Cells ,1103 Clinical Sciences ,QR Microbiology ,C500 ,spondyloarthritis ,QR ,Activating Transcription Factor 6 ,Arthritis & Rheumatology ,reactive arthritis ,1107 Immunology ,Salmonella Infections ,RC Internal medicine ,Unfolded Protein Response ,HLA-B35 Antigen ,BDC ,RC - Abstract
A.N.A was funded by ARUK Fellowships Non-Clinical Career Development Fellowship Ref No: 18440. I.L. was funded by an ARUK PhD studentship Ref No: 17868. A.N.A and S.J.P were also in part funded by ARUK (grant 21261) Objective Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. Methods Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. Results S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. Conclusions HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention. Publisher PDF
- Published
- 2018
4. Monocistronic high titre vectors for targeted expression of multiple therapeutic genes
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Darling, D, Gaken, J, Nesbeth, D, Chan, L, Hardwick, N, Mackey, T, Galea-Lauri, J, Vyakarnam, A, Mufti, G, Kemeny, M, and Farzaneh, F
- Published
- 2016
5. High titre vectors for targeted expression of multiple genes
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Darling, D, Gaken, J, Nesbeth, D, Chan, L, Hardwick, N, Mackey, T, Galea-Lauri, J, Vyakarnam, A, Mufti, GJ, Kemeny, M, and Farzaneh, F
- Published
- 2016
6. Step change in the efficiency of centrifugation through cell engineering: co‐expression of Staphylococcal nuclease to reduce the viscosity of the bioprocess feedstock
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Balasundaram, B., primary, Nesbeth, D., additional, Ward, J.M., additional, Keshavarz‐Moore, E., additional, and Bracewell, D.G., additional
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- 2009
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7. Affinity recovery of Moloney Murine Leukaemia Virus
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WILLIAMS, S, primary, NESBETH, D, additional, DARLING, D, additional, FARZANEH, F, additional, and SLATER, N, additional
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- 2005
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8. Novel detection of in vivo HLA-B27 conformations correlates with ankylosing spondylitis association.
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Fussell H, Nesbeth D, Lenart I, Campbell EC, Lynch S, Santos S, Gould K, Powis SJ, and Antoniou AN
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OBJECTIVE: The class I major histocompatibility complex (MHC) molecule HLA-B27 exhibits a strong association with the autoimmune inflammatory arthritis disorder ankylosing spondylitis (AS) and with other related spondylarthropathies. In the absence of both a defined autoimmune response and a target autoantigen(s), the propensity of HLA-B27 to misfold has been hypothesized to be a major parameter in disease pathogenesis. We undertook this study to test the hypothesis that HLA-B27 misfolding is due to exposure of cysteine residues within the heavy chain to the oxidizing environment of the endoplasmic reticulum. METHODS: A rapid acidification and alkylation modification method was used to examine cysteine residue exposure and accessibility within AS-associated and non-AS-associated HLA-B27 subtypes. RESULTS: This novel approach to probing in vivo class I MHC structure revealed that the HLA-B27 heavy chain adopts conformations not previously described. Furthermore, amino acid residues specific to subtypes HLA-B*2706, B*2709, and B*2704 can have an impact on these novel conformations and on cysteine residue exposure. CONCLUSION: HLA-B27 can adopt novel conformations, resulting in differential accessibility of cysteine residues, which can explain the propensity to misfold. Cysteine exposure in the HLA-B27 heavy chain is also affected by residues within the 114 and 116 regions, thereby providing a potential biochemical basis for the association of HLA-B27 subtypes with AS. [ABSTRACT FROM AUTHOR]
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- 2008
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9. Development of a xeno-free cell culture platform for novel candidate human cell therapies for neural regeneration
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Santiago Toledo, Gerardo, Wall, I., Jat, P. S., Nesbeth, D., and Roberton, V.
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660.6 - Abstract
The olfactory mucosa is a source of cell types that are associated with neural regeneration such as olfactory ensheathing cells (OECs), mesenchymal stromal cells (MSCs), and neural stem cells (NSCs). Although cells from this region have been used for autologous transplantation onto damaged spinal cord injury, there is a gap for an allogeneic cell therapy approach. The aim of this thesis was to establish key bioprocessing parameters for the expansion of c-MycERTAM-derived populations of human late-adherent olfactory mucosa cells (hOMCs). First, growth kinetics, identity and potency were characterised in both monolayer and suspension culture platforms. Profiles of cell growth kinetics were obtained for seeding densities ranging from 3,000 cells/cm² to 12,000 cells/cm² by manual counting and confluence measurements. PA5 hOMCs achieved between 35-41 population doublings in culture when seeded at 6,000 cells/cm² in long term culture. Moreover, removal of 4-hydroxytamoxifen (4-OHT) did not have an effect on cell growth kinetics. Replicative senescence was observed due to undesired silencing of c-MycERTAM. Next, alternative media compositions were examined as a means of optimising the process and transitioning to xeno-free culture. hOMCs showed a high dependence on fetal bovine serum (FBS) supplementation, and substitution with commercially available supplements did not sustain growth in serum-free conditions. However, human platelet lysate (hPL) was found to be a comparable to FBS when used to supplement basal media at concentrations of 2% and 5% (v/v). In this xeno-free media, hOMCs showed comparable cell growth kinetics to FBS supplemented media. Additionally, identity and potency marker expression in hOMCs was comparable between the FBS and the xeno-free hPL conditions. Finally, to progress towards scalable production of hOMCs, microcarrier screening and subsequent cell expansion on microcarriers in spinner flasks was undertaken. PA5 hOMCs were successfully expanded as a suspension culture on microcarriers at 80-mL scale in spinner flasks. A total of 8.40 ± 0.54 ×10⁶ viable cells were harvested when grown in medium supplemented with 5% hPL, and a similar number of 6.70 ± 1.05 ×10⁶ cells when grown in 10% FBS using Plastic microcarriers. PA5 hOMCs expanded on microcarriers conserved markers of identity post-harvest. In conclusion, by addressing bioprocessing fundamentals, advances towards scalable production of hOMCs using xeno-free culture reagents have been achieved.
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- 2019
10. Improved production and purification of recombinant proteins from mammalian expression systems
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Kinna, A. W., Nesbeth, D., and Chester, K. A.
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660.6 - Abstract
The biopharmaceutical industry is becoming increasingly reliant on recombinant proteins as therapeutic agents. The work presented here details the development of rapid mammalian expression systems and novel capture methods for use in early recombinant protein development. A key aim was to investigate expression of recombinant proteins via cost effective production methods and to compare the resultant products at small scales of manufacture. A model single chain variable fragment Fc conjugate (scFv-Fc) targeted against a clinically relevant glycoprotein, the carcinoembryonic antigen (CEA), was expressed and characterised using both transient and stable-based expression of transgenic DNA. Transient gene expression in suspension HEK293 cells produced a maximum scFv-Fc level of 72μg/mL, which was used for initial protein characterisation. A stable pool of transfected CHO cells was also generated using a ubiquitous chromatin opening element (UCOE)-based vector achieving increased protein expression and culture periods when combined with a fed-batch regimen. Characterisation of the resulting proteins showed that stability and effector function was maintained across transient and stable production methods at all scales, indicating that preliminary data generated from transient expression in HEK293F cells could be generalised to predict that of protein stably expressed in CHO cell populations. A significant bottleneck in harvesting and purifying proteins from cell containing feed streams is the requirement for solid-liquid separation prior to capture. Therefore, a technique was proposed for direct capture of recombinant protein from unclarified feed streams that can integrate directly into the bioreactor harvest line. The process was demonstrated using immobilised metal affinity chromatography (IMAC) capture of recombinant CEA with a polyhistidine (His6) tag from a bioreactor culture. This provides a basis for direct primary capture of recombinant proteins from unclarified mammalian cell feed streams that could be generalized to other capture methods and proteins.
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- 2017
11. Industrially robust synthetic biology standards for the polymerase chain reaction
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Templar, A. and Nesbeth, D.
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660.6 - Abstract
Synthetic Biology is ushering in a new era where reengineered genomes can enhance the capacity of host cells to produce biologic and chemical products. Standardisation is a key component of synthetic biology as it enables effective implementation (Müller and Arndt, 2012). This project has successfully generated synthetic biology standards for the quantitative polymerase chain reaction (qPCR), a highly specific and sensitive analytical platform, in order to increase its robustness for monitoring of host cell processes in an industrial setting. This project has also increased the assay throughput to allow for at-line analysis, in accordance with initiatives such as Process Analytical Technology (PAT) (Gnoth et al., 2007). Analysis was conducted on three commonly used host cell chassis and industrial contamination was also simulated by the addition of plasmid proxies. All assays were optimised by primer design and screening to ensure accuracy. End point PCR (e -pPCR) and quantitative PCR (qPCR) was conducted in the presence and absence of cellular material disrupted by a mild sonication procedure. We found that, whilst cellular material reduces assay sensitivity for a genomic locus, the presence of contaminating species can be accurately quantified. We also employed LRE-qPCR, which uses the CAL1 standard for quantification. LRE-qPCR matched the accuracy of a conventional standard curve qPCR method and we propose it as a Synthetic Biology standard. We next developed a modified standard curve method that streamlined methodology and bypassed errors inherent to the gold standard methodology to, for the first time, enable quantification of multiple targets from a single standard curve. The CyCal curve is a standard curve constructed from the CAL1 standard combined with the Cy0 data analysis. The approach was validated against 6 bioprocess targets and it was found that CyCal was able to replicate the accuracy of the gold standard approach. We then used CyCal to accurately determine how host cell plasmid copy number (PCN) evolves during fermentation. The combination of rapid sample preparation and a universal standard means that CyCal is capable of becoming the basis of an at-line qPCR assay when conducted on modern ultra-rapid qPCR thermocycler technology.
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- 2017
12. Synthetic biology approaches to bioprocess intensification in bacterial and mammalian production platforms
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Schofield, D. M. and Nesbeth, D.
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660.6 - Abstract
The controlled production of recombinant proteins and their high purity recovery are key goals of biochemical engineering, and can be met by concerted design of cellular hosts using synthetic biology tools. This thesis addresses five process areas with biological solutions. DNA is a major impurity in mammalian process streams, and can be removed by adding nuclease to the feedstream. Here, a HeLa cell line which encodes a recombinant nuclease is successfully adapted to serum-free growth and its activity is characterised. Optimising protein induction within E. coli is a time consuming process; here a synthetic gene network which is predicted to continually produce a recombinant protein once activated is assessed and the host cell line, culture environment, and analytical tools developed. Previously work with a biotherapeutic protein had failed to produce a dimer in a high yield or pure form from E. coli inclusion bodies. A cysteine residue was substituted into the sequence for dimerisation, and a bench scale production process yielding 108 mg of 95% pure dimer was developed. The monomeric form was chemically crosslinked to produce an enhanced stability dimer. DNA increases homogenate viscosity; here, an E. coli strain expressing a SRP-trafficked nuclease is constructed and transformed with a plasmid encoding an SEC-trafficked antibody fragment (Fab'); this strain autohydrolyses DNA, but maintained a high Fab' yield. This contrasts with a previously constructed SEC-trafficked nuclease, which reduced the yield and increased cell lysis. Proteomic analysis showed that utilising two translocation pathways depleted periplasmic proteins and a deterministic model suggested a maximum of 6% of the total periplasm volume could be occupied by recombinant proteins, above which lysis occurred. In order to test this threshold, a promoter-engineered variant of the Fab' production strain was constructed. Bench-scale fermentations showed improved product retention, cell viability, and validated the 6% hypothesis.
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- 2016
13. Design, mathematical modelling, construction and testing of synthetic gene network oscillators to establish Roseobacter clade bacteria and the protozoan Trypanosoma brucei as synthetic biology chassis
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Borg, Y., Nesbeth, D., and Zaikin, A.
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660.6 - Abstract
The aim of this project is to establish Roseobacter marine bacteria and Trypanosoma brucei (T. brucei) protozoa as synthetic biology chassis. This work addresses the gap within synthetic biology resulting from the limited choice of host cells available for use in practice. This was done by developing synthetic bacterial and trypanosomal genetic regulatory networks (GRNs) which function as an oscillator as well as by developing the necessary protocols and set-ups to allow for the analysis of GRN dynamics within the host. Roseobacter clade bacteria are naturally found in diverse oceanic habitats and have an important ecological role in balancing global carbon levels. This makes Roseobacter an ideal chassis for future efforts to apply synthetic biology to bioremediation and geo-engineering challenges. The aim of this investigation was to establish straight-forward molecular biology procedures in Roseobacter bacteria followed by characterisation and modelling of an E. coli oscillator in Roseobacter. Results showed that Roseobacter synthetic biology is non-trivial. Protozoa are exploited as host cells for industrial production of biotherapeutics due to fast doubling times and host proteins' mammalian-like post-translational glycosylation. As an established model organism for studying protozoa, T. brucei provided a test case for establishing synthetic biology in this phylum for the first time. T. brucei is highly divergent from eukaryotes commonly used in synthetic biology and possesses a sophisticated genomic machinery to evade host immune systems. The establishment of standard synthetic biology approaches in mathematical modelling and gene network design in T. brucei will underpin application of synthetic biology to enhance the industrial capability of the protozoa as a chassis and to probe its pathobiology. This investigation involved design and assembly of a Goodwin oscillator, followed by characterisation and modelling of the network and the development of a novel experimental set-up for live-cell imaging of single motile trypanosomes. Results showed that T. brucei is a promising novel synthetic biology chassis.
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- 2015
14. Identification of black sturgeon caviar pigment as eumelanin
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Marco d'Ischia, Gerardino D'Errico, Alessandra Napolitano, Darren N. Nesbeth, Kenneth Benning, Brunella Setaro, Lucia Panzella, Panzella, L., Benning, K., Nesbeth, D. N., Setaro, B., D'Errico, G., Napolitano, A., and D'Ischia, M.
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Sturgeon roe ,Oxidative degradation ,Food chemistry ,Mass Spectrometry ,Analytical Chemistry ,law.invention ,Melanin ,Pigment ,Sturgeon ,Electron paramagnetic resonance (EPR) ,law ,Animals ,Sepia ,Electron paramagnetic resonance ,Chromatography, High Pressure Liquid ,Melanins ,Chromatography ,Black caviar ,Animal ,Chemistry ,Pigmentation ,General Medicine ,visual_art ,visual_art.visual_art_medium ,%22">Fish ,Pyrrole-2,3,5-tricarboxylic acid (PTCA) ,Food Science - Abstract
Reported herein is the purification of the pigment of black sturgeon caviar and its unambiguous identification as a typical eumelanin by means of chemical degradation coupled with electron paramagnetic resonance (EPR) evidence. HPLC and LC-MS analysis of oxidative degradation mixtures revealed the formation of pyrrole-2,3,5-tricarboxylic acid (PTCA), a specific marker of eumelanin pigments, in yields compatible with a 6.5% w/w pigment content. EPR spectral features and parameters were in close agreement with those reported for a typical natural eumelanin such as Sepia melanin from squid ink. The identification for the first time of eumelanin in a fish roe is expected to provide a novel molecular basis for the valorization of black caviar and production wastes thereof in food chemistry and diet.
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- 2021
15. 601 - Gene Editing/Gene Therapies: ENGINEERING HEK293T CELL LINES TO EXPRESS NUCLEASE FOR DNA CLEARANCE IN LENTIVIRAL VECTOR PRODUCTION.
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Ali, S., Rivera, M., Ward, J., Keshavarz-Moore, E., Mason, C., Qasim, W., and Nesbeth, D.
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- *
GENOME editing , *GENE therapy , *CELL lines , *DNA , *ENGINEERING - Published
- 2022
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16. Salmonella Exhibit Altered Cellular Localization in the Presence of HLA-B27 and Codistribute with Endo-Reticular Membrane.
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Kriston-Vizi J, Lenart I, Iwawaki T, Gould K, Nesbeth D, Powis SJ, and Antoniou AN
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- Cell Line, Epithelial Cells, Humans, Salmonella metabolism, HLA-B27 Antigen genetics, HLA-B27 Antigen metabolism, Salmonella Infections
- Abstract
Salmonella enteritica ( S. enteritica ) induce and require unfolded protein response (UPR) pathways for intracellular replication. Salmonella infections can lead to reactive arthritis (ReA), which can exhibit associations with Human Leucocyte Antigen (HLA)-B
∗ 27 : 05. S. enteritica normally reside in a juxtanuclear position to the Golgi apparatus, representing the formation and residence within the Salmonella -containing vacuole (SCV). Changes in cellular localization of infecting Salmonella can alter their ability to replicate. We therefore used isogenic epithelial cell lines expressing physiological levels of HLA-B∗ 27 : 05 heavy chain (HC) and a control HLA-B allele, HLA-B∗ 35 : 01.HC to determine any changes in Salmonella localization within epithelial cells. Expression of HLA-B∗ 27 : 05 but not HLA-B∗ 35 : 01 was associated with a quantifiable change in S. enteritica cellular distribution away from the Golgi apparatus. Furthermore, the Salmonella requirements for UPR induction and the consequences of the concomitant endoplasmic reticulum (ER) membrane expansion were determined. Using confocal imaging, S. enteritica bacteria exhibited a significant and quantifiable codistribution with endo-reticular membrane as determined by ER tracker staining. Isogenic S. enterica Typhimurium mutant strains, which can infect but exhibit impaired intracellular growth, demonstrated that the activation of the UPR was dependent on an integral intracellular niche. Therefore, these data identify cellular changes accompanying Salmonella induction of the UPR and in the presence of HLA-B27., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Janos Kriston-Vizi et al.)- Published
- 2022
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17. Intrinsic Folding Properties of the HLA-B27 Heavy Chain Revealed by Single Chain Trimer Versions of Peptide-Loaded Class I Major Histocompatibility Complex Molecules.
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Lenart I, Truong LH, Nguyen DD, Rasiukienė O, Tsao E, Armstrong J, Kumar P, McHugh K, Pereira BI, Maan BS, Garstka MA, Bowness P, Blake N, Powis SJ, Gould K, Nesbeth D, and Antoniou AN
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- Antigen Presentation, Genes, MHC Class I, Humans, Molecular Chaperones genetics, Peptides genetics, HLA-B27 Antigen genetics, Histocompatibility Antigens Class I genetics
- Abstract
Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (β2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lenart, Truong, Nguyen, Rasiukienė, Tsao, Armstrong, Kumar, McHugh, Pereira, Maan, Garstka, Bowness, Blake, Powis, Gould, Nesbeth and Antoniou.)
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- 2022
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18. A Systematic Review of the Criminogenic Potential of Synthetic Biology and Routes to Future Crime Prevention.
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Elgabry M, Nesbeth D, and Johnson SD
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Synthetic biology has the potential to positively transform society in many application areas, including medicine. In common with all revolutionary new technologies, synthetic biology can also enable crime. Like cybercrime, that emerged following the advent of the internet, biocrime can have a significant effect on society, but may also impact on peoples' health. For example, the scale of harm caused by the SARS-CoV-2 pandemic illustrates the potential impact of future biocrime and highlights the need for prevention strategies. Systematic evidence quantifying the crime opportunities posed by synthetic biology has to date been very limited. Here, we systematically reviewed forms of crime that could be facilitated by synthetic biology with a view to informing their prevention. A total of 794 articles from four databases were extracted and a three-step screening phase resulted in 15 studies that met our threshold criterion for thematic synthesis. Within those studies, 13 exploits were identified. Of these, 46% were dependent on technologies characteristic of synthetic biology. Eight potential crime types emerged from the studies: bio-discrimination, cyber-biocrime, bio-malware, biohacking, at-home drug manufacturing, illegal gene editing, genetic blackmail, and neuro-hacking. 14 offender types were identified. For the most commonly identified offenders (>3 mentions) 40% were outsider threats. These observations suggest that synthetic biology presents substantial new offending opportunities. Moreover, that more effective engagement, such as ethical hacking, is needed now to prevent a crime harvest from developing in the future. A framework to address the synthetic biology crime landscape is proposed., (Copyright © 2020 Elgabry, Nesbeth and Johnson.)
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- 2020
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19. A systematic review protocol for crime trends facilitated by synthetic biology.
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Elgabry M, Nesbeth D, and Johnson SD
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- Computer Security, Genetic Engineering, Humans, Synthetic Biology, Systematic Reviews as Topic, Biotechnology standards, Crime, Security Measures standards
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Background: When new technologies are developed, it is common for their crime and security implications to be overlooked or given inadequate attention, which can lead to a 'crime harvest'. Potential methods for the criminal exploitation of biotechnology need to be understood to assess their impact, evaluate current policies and interventions and inform the allocation of limited resources efficiently. Recent studies have illustrated some of the security implications of biotechnology, with outcomes of misuse ranging from compromised computers using malware stored in synthesised DNA, infringement of intellectual property on biological matter, synthesis of new threatening viruses, 'genetic genocide,' and the exploitation of food markets with genetically modified crops. However, there exists no synthesis of this information, and no formal quality assessment of the current evidence. This review therefore aims to establish what current and/or predicted crimes have been reported as a result of biotechnology., Methods: A systematic review will be conducted to identify relevant literature. ProQuest, Web of Science, MEDLINE and USENIX will be searched utilizing a predefined search string, and Backward and Forward searches. Grey literature will be identified by searching the official UK Government website (www.gov.uk) and the Global database of Dissertations and Theses. The review will be conducted by screening title/abstracts followed by full texts, utilising pre-defined inclusion and exclusion criteria. Papers will be managed using Eppi-center Reviewer 4 software, and data will be organised using a data extraction table using a descriptive coding tool. A predefined rating system (speculative, experimental or currently occurring) will be used to sort studies, and a thematic synthesis of the results will be presented., Discussion: Despite the concerns raised about the misuse of biotechnology, no previous work has been conducted from a Crime Science perspective to collate and assess the literature. This systematic review aims to identify the types of offending activity facilitated by biotechnology, including synthetic biology and genetic engineering. The objective of the review is to examine whether this offending activity can be prevented by assessing the conditions necessary for the crime events to occur. It is anticipated that evidence generated from this review will guide future research in this area and aid relevant stakeholders to prioritise and allocate limited resources to biotechnology crime prevention., Systematic Review Registration: PROSPERO CRD42019131685.
- Published
- 2020
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20. Sorbitol/methanol mixed induction reduces process impurities and improves centrifugal dewatering in Pichia pastoris culture.
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Wang B, Nesbeth D, and Keshavarz-Moore E
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- Biomass, Cell Survival, Fermentation, Oxygen metabolism, Pichia growth & development, Recombinant Proteins, Centrifugation, Methanol metabolism, Pichia metabolism, Sorbitol metabolism
- Abstract
This study investigates how sorbitol/methanol mixed induction affects fermentation performance, dewatering characteristics of cells during harvesting and the profile of host cell proteins (HCP) in the process fluid when producing the target recombinant protein aprotinin. Compared to standard methanol induction, sorbitol/methanol (1:1, C-mol/C-mol) mixed induction improved cellular viability from 92.8 ± 0.3% to 97.7 ± 0.1% although resulted in a reduced product yield from 1.65 ± 0.03 g L
-1 to 1.12 ± 0.07 g L-1 . On the other hand, average oxygen consumption rate (OUR) dropped from 241.4 ± 21.3 mmol L-1 h-1 to 145.5 ± 6.7 mmol L-1 h-1 . Cell diameter decreased over time in the mixed induction, resulting in a D50 value of 3.14 μm at harvest compared to 3.85 μm with methanol. The reduction in cell size enhanced the maximum dewatering efficiency from 78.1 ± 3.9% to 84.5 ± 3.3% as evaluated by using an established ultra scale-down methodology that models pilot and industrial scale disc stack centrifugation. Seventy host cell proteins (HCPs) were identified in clarified supernatant when using sorbitol/methanol mixed induction regimen. The total number of HCPs identified with standard methanol induction was nearly one hundred. The downstream process advantage of the mixed induction lies in improved product purity by reducing both cell mortality and level of released whole cell proteins. This needs to be balanced and optimised against the observed reduction in product yield during fermentation., (Copyright © 2019. Published by Elsevier Inc.)- Published
- 2019
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21. Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy.
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Folarin O, Nesbeth D, Ward JM, and Keshavarz-Moore E
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There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of -0.022, and pSVβ-Gal -0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.
- Published
- 2019
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22. Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication.
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Antoniou AN, Lenart I, Kriston-Vizi J, Iwawaki T, Turmaine M, McHugh K, Ali S, Blake N, Bowness P, Bajaj-Elliott M, Gould K, Nesbeth D, and Powis SJ
- Subjects
- Activating Transcription Factor 6 metabolism, Arthritis, Reactive microbiology, Cell Cycle, Cell Line, HLA-B35 Antigen metabolism, Humans, Prohibitins, Salmonella Infections complications, X-Box Binding Protein 1 metabolism, Epithelial Cells cytology, HLA-B27 Antigen metabolism, Salmonella Infections microbiology, Salmonella enterica metabolism, Unfolded Protein Response physiology
- Abstract
Objective: Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells., Methods: Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane., Results: S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism., Conclusions: HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)
- Published
- 2019
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23. Biotransformation of β-hydroxypyruvate and glycolaldehyde to l-erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase.
- Author
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Wei YC, Braun-Galleani S, Henríquez MJ, Bandara S, and Nesbeth D
- Subjects
- Acetaldehyde chemistry, Bioreactors, Fermentation, Gene Expression Regulation, Fungal, Methanol chemistry, Pichia chemistry, Pichia genetics, Promoter Regions, Genetic, Recombinant Proteins chemistry, Recombinant Proteins genetics, Tetroses biosynthesis, Transketolase chemistry, Transketolase genetics, Acetaldehyde analogs & derivatives, Biotransformation, Pyruvates chemistry, Tetroses chemistry
- Abstract
Transketolase is a proven biocatalytic tool for asymmetric carbon-carbon bond formation, both as a purified enzyme and within bacterial whole-cell biocatalysts. The performance of Pichia pastoris as a host for transketolase whole-cell biocatalysis was investigated using a transketolase-overexpressing strain to catalyze formation of l-erythrulose from β-hydroxypyruvic acid and glycolaldehyde substrates. Pichia pastoris transketolase coding sequence from the locus PAS_chr1-4_0150 was subcloned downstream of the methanol-inducible AOX1 promoter in a plasmid for transformation of strain GS115, generating strain TK150. Whole and disrupted TK150 cells from shake flasks achieved 62% and 65% conversion, respectively, under optimal pH and methanol induction conditions. In a 300 μL reaction, TK150 samples from a 1L fed-batch fermentation achieved a maximum l-erythrulose space time yield (STY) of 46.58 g L
-1 h-1 , specific activity of 155 U gCDW-1, product yield on substrate (Yp/s ) of 0.52 mol mol-1 and product yield on catalyst (Yp/x ) of 2.23g gCDW-1. We have successfully exploited the rapid growth and high biomass characteristics of Pichia pastoris in whole cell biocatalysis. At high cell density, the engineered TK150 Pichia pastoris strain tolerated high concentrations of substrate and product to achieve high STY of the chiral sugar l-erythrulose. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:99-106, 2018., (© 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.)- Published
- 2018
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24. IMAC capture of recombinant protein from unclarified mammalian cell feed streams.
- Author
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Kinna A, Tolner B, Rota EM, Titchener-Hooker N, Nesbeth D, and Chester K
- Subjects
- Animals, CHO Cells metabolism, Carcinoembryonic Antigen isolation & purification, Carcinoembryonic Antigen metabolism, Cell Survival, Cricetulus, Microspheres, Recombinant Fusion Proteins metabolism, Sodium Chloride metabolism, Chromatography, Affinity methods, Recombinant Fusion Proteins isolation & purification
- Abstract
Fusion-tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300-500 μm diameter agarose resin beads that allow free passage of cells but capture His-tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His-tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼ 8 U/mL and 2 ng/μL in column flow-through, respectively. Recovery of His-tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams., (© 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)
- Published
- 2016
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25. Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.
- Author
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Guiliano DB, Fussell H, Lenart I, Tsao E, Nesbeth D, Fletcher AJ, Campbell EC, Yousaf N, Williams S, Santos S, Cameron A, Towers GJ, Kellam P, Hebert DN, Gould KG, Powis SJ, and Antoniou AN
- Subjects
- DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins drug effects, DNA-Binding Proteins physiology, HeLa Cells, Humans, Membrane Proteins antagonists & inhibitors, Membrane Proteins drug effects, RNA, Small Interfering pharmacology, Regulatory Factor X Transcription Factors, Signal Transduction physiology, Transcription Factors antagonists & inhibitors, Transcription Factors drug effects, Transcription Factors physiology, Ubiquitin-Protein Ligases antagonists & inhibitors, Ubiquitin-Protein Ligases drug effects, Ubiquitin-Protein Ligases physiology, X-Box Binding Protein 1, Endoplasmic Reticulum physiology, Endoplasmic Reticulum-Associated Degradation physiology, HLA-B27 Antigen physiology, Membrane Proteins physiology, Protein Folding
- Abstract
Objective: HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers., Methods: HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays., Results: We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2., Conclusion: The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease., (Copyright © 2014 by the American College of Rheumatology.)
- Published
- 2014
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26. Rapid acidification and alkylation: redox analysis of the MHC class I pathway.
- Author
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Powis SJ, Nesbeth D, Lenart I, Fussell H, Lamb T, Gould K, and Antoniou AN
- Subjects
- Alkylation, Cysteine chemistry, Cysteine immunology, Cysteine metabolism, HLA-A2 Antigen chemistry, HLA-A2 Antigen immunology, HLA-B27 Antigen chemistry, HLA-B27 Antigen immunology, Humans, Hydrogen-Ion Concentration, Oxidation-Reduction, Oxidoreductases chemistry, Oxidoreductases immunology, Stilbenes chemistry, Sulfonic Acids chemistry, Trichloroacetic Acid chemistry, HLA-A2 Antigen metabolism, HLA-B27 Antigen metabolism, Oxidoreductases metabolism
- Abstract
The technique of rapid acidification and alkylation can be used to characterise the redox status of oxidoreductases, and to determine numbers of free cysteine residues within substrate proteins. We have previously used this method to analyse interacting components of the MHC class I pathway, namely ERp57 and tapasin. Here, we have applied rapid acidification/alkylation as a novel approach to analysing the redox status of MHC class I molecules. This analysis of the redox status of the MHC class I molecules HLA-A2 and HLA-B27, which is strongly associated with a group of inflammatory arthritic disorders referred to as Spondyloarthropathies, revealed structural and conformational information. We propose that this assay provides a useful tool in the study of in vivo MHC class I structure.
- Published
- 2009
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27. Metabolic biotinylation of lentiviral pseudotypes for scalable paramagnetic microparticle-dependent manipulation.
- Author
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Nesbeth D, Williams SL, Chan L, Brain T, Slater NK, Farzaneh F, and Darling D
- Subjects
- Biotin metabolism, Carbon-Nitrogen Ligases metabolism, Cell Line, Endoplasmic Reticulum metabolism, Escherichia coli Proteins metabolism, Genetic Vectors genetics, Humans, Lentivirus genetics, Models, Biological, Moloney murine leukemia virus genetics, Receptor, Nerve Growth Factor metabolism, Repressor Proteins metabolism, Transcription Factors metabolism, Vesicular stomatitis Indiana virus genetics, Viral Proteins genetics, Viral Proteins metabolism, Biotinylation, Gene Transfer Techniques, Genetic Vectors physiology, Lentivirus physiology, Magnetics, Microspheres
- Abstract
Nonviral, host-derived proteins on lentiviral vector surfaces can have a profound effect on the vector's biology as they can both promote infection and provide resistance to complement inactivation. We have exploited this to engineer a specific posttranslational modification of a "nonenvelope," virally associated protein. The bacterial biotin ligase (BirA) and a modified human DeltaLNGFR have been introduced into HEK293T cells and their protein products directed to the lumen of the endoplasmic reticulum. The BirA then couples biotin to an acceptor peptide that has been fused to the DeltaLNGFR. This results in the covalent linkage of biotin to the extracellular domain of the DeltaLNGFR expressed on the cell surface. Lentiviral vectors from these cells are metabolically labeled with biotin in the presence of free biotin. These biotinylated lentiviral vectors have a high affinity for streptavidin paramagnetic particles and, once captured, are easily manipulated in vitro. This is illustrated by the concentration of lentiviral vectors pseudotyped with either the VSV-G or an amphotropic envelope in excess of 4500-fold. This new cell line has the potential for widespread application to envelope pseudotypes compatible with lentiviral vector production.
- Published
- 2006
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28. Conjugation of lentivirus to paramagnetic particles via nonviral proteins allows efficient concentration and infection of primary acute myeloid leukemia cells.
- Author
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Chan L, Nesbeth D, Mackey T, Galea-Lauri J, Gäken J, Martin F, Collins M, Mufti G, Farzaneh F, and Darling D
- Subjects
- Cell Line, GTP-Binding Proteins genetics, GTP-Binding Proteins metabolism, Genetic Vectors genetics, Humans, Lentivirus genetics, Tumor Cells, Cultured virology, Vesicular stomatitis Indiana virus chemistry, Virus Replication, Gene Transfer Techniques, Genetic Vectors physiology, Lentivirus physiology, Leukemia, Myeloid, Acute, Magnetics, Membrane Proteins metabolism, Microspheres
- Abstract
Nonviral producer cell proteins incorporated into retroviral vector surfaces profoundly influence infectivity and in vivo half-life. We report the purification and concentration of lentiviral vectors using these surface proteins as an efficient gene transduction strategy. Biotinylation of these proteins and streptavidin paramagnetic particle concentration enhances titer 400- to 2,500-fold (to 10(9) CFU/ml for vesicular stomatitis virus G protein and 5 x 10(8) for amphotropic murine leukemia virus envelope). This method also uses newly introduced membrane proteins (B7.1 and DeltaLNGFR) directed to lentiviral surfaces, allowing up to 17,000-fold concentrations. Particle conjugation of lentivirus allows facile manipulation in vitro, resulting in the transduction of 48 to 94% of human acute myeloid leukemia blasts.
- Published
- 2005
- Full Text
- View/download PDF
29. Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition.
- Author
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Wilbourn B, Nesbeth DN, Wainwright LJ, and Field MC
- Subjects
- Acetylcysteine analogs & derivatives, Acetylcysteine pharmacology, Animals, CHO Cells, Cricetinae, Cysteine metabolism, Cysteine Endopeptidases metabolism, Cysteine Proteinase Inhibitors pharmacology, Endoplasmic Reticulum physiology, Glycosylphosphatidylinositols genetics, Human Growth Hormone chemistry, Human Growth Hormone genetics, Humans, Leupeptins pharmacology, Multienzyme Complexes metabolism, Mutation genetics, Peptides pharmacology, Proteasome Endopeptidase Complex, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Ubiquitins metabolism, Glycosylphosphatidylinositols metabolism, Sulfhydryl Compounds metabolism
- Abstract
Improperly processed secretory proteins are degraded by a hydrolytic system that is associated with the endoplasmic reticulum (ER) and appears to involve re-export of lumenal proteins into the cytoplasm for ultimate degradation by the proteasome. The chimaeric protein hGHDAF28, which contains a crippled glycosylphosphatidylinositol (GPI) C-terminal signal peptide, is degraded by a pathway highly similar to that for other ER-retained proteins and is characterized by formation of disulphide-linked aggregates, failure to reach the Golgi complex and intracellular degradation with a half life of approximately 2 h. Here we show that N-acetyl-leucinal-leucinal-norleucinal, MG-132 and lactacystin, all inhibitors of the proteasome, protect hGHDAF28; hGHDAF28 is still proteolytically cleaved in the presence of lactacystin or MG-132, by the removal of approximately 2 kDa, but the truncated fragment is not processed further. We demonstrate that the ubiquitination system accelerates ER-degradation of hGHDAF28, but is not essential to the process. Overall, these findings indicate that GPI quality control is mediated by the cytoplasmic proteasome. We also show that the presence of a cysteine residue in the GPI signal of hGHDAF28 is required for retention and degradation, as mutation of this residue to serine results in secretion of the fusion protein, implicating thiol-mediated retention as a mechanism for quality control of some GPI signals. Removal of the cysteine also prevents inclusion of hGHDAF28 in disulphide-linked aggregates, indicating that aggregate formation is an additional retention mechanism for this class of protein. Therefore our data suggest that an unpaired terminal cysteine is the retention motif of the hGHDAF28 GPI-processing signal and that additional information may be required for efficient engagement of ER quality control systems by the majority of GPI signals which lack cysteine residues.
- Published
- 1998
- Full Text
- View/download PDF
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