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  • 1
    Language: English
    In: Bioanalysis, January 2013, Vol.5(2), pp.123-6
    Keywords: Biopharmaceutics -- Trends ; Drug Industry -- Trends
    ISSN: 17576180
    E-ISSN: 1757-6199
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  • 2
    Language: English
    In: PLoS ONE, 2011, Vol.6(12), p.e28271
    Description: Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems. ; As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state. ; Our results support the hypothesis that PhLP3 is important for the maintenance of β-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth.
    Keywords: Research Article ; Biology ; Engineering ; Molecular Biology ; Biotechnology ; Cell Biology ; Biochemistry
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: Journal of Chromatography A, 26 August 2016, Vol.1461, pp.70-77
    Description: Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC–MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC–MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.
    Keywords: Protein A Affinity Chromatography ; Host Cell Proteins ; LC–MS/MS ; Resin Ageing ; Process-Related Impurities ; Bioprocessing ; Chemistry
    ISSN: 0021-9673
    E-ISSN: 18733778
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  • 4
    Language: English
    In: Biotechnology and Bioengineering, 15 February 2010, Vol.105(3), pp.556-566
    Description: Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co‐expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over‐expression of the yeast ‘protein disaggregase’ Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over‐expression of the yeast or mammalian Hsp70/40 chaperones. Over‐expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5‐fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over‐expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over‐expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells. Biotechnol. Bioeng. 2010; 105: 556–566. © 2009 Wiley Periodicals, Inc.
    Keywords: Molecular Chaperone ; Hsp104 ; Protein Secretion ; Luciferase ; Protein Disaggregation ; Torsina
    ISSN: 0006-3592
    E-ISSN: 1097-0290
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  • 5
    Language: English
    In: Biotechnology and Bioengineering, January 2013, Vol.110(1), pp.240-251
    Description: Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D‐PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post‐protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D‐PAGE can be used for monitoring and identification of HCPs post‐protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell‐engineering approaches can be applied to reduced, or eliminate, such HCPs. Biotechnol. Bioeng. 2013; 110: 240–251. © 2012 Wiley Periodicals, Inc. A proteomics analysis approach (2D‐PAGE) has revealed that subsequent downstream processing choices following the harvest of the cell culture supernatant from Chinese hamster ovary cell lines expressing a model monoclonal antibody impacts upon the host cell protein (HCP) profile. We utilized an ultra scale down approach which could be used in early process development to aid the design/selection of clarification techniques to minimize HCP content and/or to screen potential cell lines to select those with overall reduced HCP profiles or of those HCPs which are problematic and challenging to remove.
    Keywords: Host Cell Protein ; Chinese Hamster Ovary Cells ; Downstream Processing ; Monoclonal Antibody ; Protein A Chromatography ; 2d‐Page
    ISSN: 0006-3592
    E-ISSN: 1097-0290
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  • 6
    Language: English
    In: Biotechnology and Bioengineering, 09/2011, Vol.108(9), pp.2246-2246
    Keywords: Engineering ; Biology ; Chemistry ; Anatomy & Physiology;
    ISSN: Biotechnology and Bioengineering
    E-ISSN: 00063592
    E-ISSN: 10970290
    Source: Wiley (via CrossRef)
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  • 7
    Language: English
    In: The Biochemical journal, 01 March 2014, Vol.458(2), pp.213-24
    Description: eIF3 (eukaryotic initiation factor 3) is the largest and most complex eukaryotic mRNA translation factor in terms of the number of protein components or subunits. In mammals, eIF3 is composed of 13 different polypeptide subunits, of which five, i.e. a, b, c, g and i, are conserved and essential in vivo from yeasts to mammals. In the present study, we show that the eukaryotic cytosolic chaperonin CCT [chaperonin containing TCP-1 (tailless complex polypeptide 1)] binds to newly synthesized eIF3b and promotes the correct folding of eIF3h and eIF3i. Interestingly, overexpression of these last two subunits is associated with enhanced translation of specific mRNAs over and above the general enhancement of global translation. In agreement with this, our data show that, as CCT is required for the correct folding of eIF3h and eIF3i subunits, it indirectly influences gene expression with eIF3i overexpression enhancing both cap- and IRES (internal ribosome entry segment)-dependent translation initiation, whereas eIF3h overexpression selectively increases IRES-dependent translation initiation. Importantly, these studies demonstrate the requirement of the chaperonin machinery for the correct folding of essential components of the translational machinery and provide further evidence of the close interplay between the cell environment, cell signalling, cell proliferation, the chaperone machinery and translational apparatus.
    Keywords: Protein Folding ; Chaperonin Containing Tcp-1 -- Physiology ; Eukaryotic Initiation Factor-3 -- Chemistry ; Protein Subunits -- Chemistry
    ISSN: 02646021
    E-ISSN: 1470-8728
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  • 8
    Language: English
    In: PLoS ONE, 01 January 2013, Vol.8(10), p.e77195
    Description: We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: 2012, Vol.7(10), p.e47422
    Description: Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.
    Keywords: Research Article ; Biology ; Immunology ; Molecular Biology ; Biotechnology ; Computational Biology
    E-ISSN: 1932-6203
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  • 10
    Language: English
    In: Molecular Biotechnology, 2013, Vol.54(2), pp.238-249
    Description: The use of a temperature shift cultivation to enhance recombinant protein yield is widely utilised in the bioprocessing industry. The responses of mammalian cells to heat stress are well characterized; however, the equivalent cold stress responses are not. In particular, the transcriptional mechanisms that lead to enhanced gene-specific expression upon cold stress have yet to be elucidated. We report here in silico and experimental identification and characterization of transcriptional control elements that regulate cold inducible RNA-binding ( CIRP ) gene expression and demonstrate these can be used for enhanced transgene expression. In silico analysis identified the core CIRP promoter and a number of conserved transcription factor-binding sites across mammalian species. The core promoter was confirmed by experimental studies that located the basal transcriptional regulatory elements of CIRP within 264 nucleotides upstream of the transcription start site. Deletion analysis of a fragment from -264 to -64 that contained two putative CAAT-binding sites abolished promoter activity. A second promoter was identified in the region -452 to -264 of the transcription start site which was able to drive transcription independent of the core promoter. As the two CIRP promoters were transcriptionally active and possibly cold responsive, we used electrophoretic mobility shift assays to show that both promoter regions are able to bind factors within a nuclear extract in a dose-dependent manner and that the formation of these complexes was specific to the promoter regions. Finally, we successfully demonstrate using a reporter gene approach that enhanced transgene expression can be achieved using the identified CIRP promoter.
    Keywords: Cold-shock ; Cold-inducible RNA-binding protein ; Promoters ; Transgene expression ; Gene expression
    ISSN: 1073-6085
    E-ISSN: 1559-0305
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