Tag-on-Demand: exploiting amber codon suppression technology for the enrichment of high-expressing membrane protein cell lines

2018 ◽  
Vol 31 (10) ◽  
pp. 389-398 ◽  
Author(s):  
Zachary T Britton ◽  
Timothy B London ◽  
Jeffrey Carrell ◽  
Bhupinder Dosanjh ◽  
Trevor Wilkinson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Lines ◽  
On Demand ◽  
Amber Codon ◽  
Amber Codon Suppression ◽  
Protein Cell

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

Antisense to the Epstein-Barr Virus (EBV)-Encoded Latent Membrane Protein 1 (LMP-1) Suppresses LMP-1 and Bcl-2 Expression and Promotes Apoptosis in EBV-Immortalized B Cells

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1721-1727 ◽  
Author(s):  
Jamie L. Kenney ◽  
Mary E. Guinness ◽  
Tyler Curiel ◽  
Jill Lacy
Keyword(s):  
Membrane Protein ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Antitumor Agents ◽  
Apoptotic Cell ◽  
Latent Membrane Protein ◽  
Therapeutic Effects ◽  
Inhibition Of Proliferation ◽  
Barr Virus ◽  
Epstein Barr

Abstract The Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP-1) is required for viral transformation and functions to protect cells from apoptotic cell death, in part, by induction of antiapoptotic genes, including Bcl-2 and A20. We have used antisense oligodeoxynucleotides targeted to LMP-1 as a strategy to suppress LMP-1 expression and thereby inhibit its functions. We have shown that levels of LMP-1 protein in EBV-positive lymphoblastoid cell lines can be reduced by in vitro treatment with unmodified oligodeoxynucleotides targeted to the first five codons of the LMP-1 open-reading frame. Furthermore, suppression of LMP-1 was associated with molecular and phenotypic effects that included downregulation of the LMP-1–inducible antiapoptotic genes, Bcl-2 and Mcl-1, inhibition of proliferation, stimulation of apoptosis, and enhancement of sensitivity to the chemotherapeutic agent, etoposide. These effects were largely sequence-specific and observed in EBV-positive, but not EBV-negative cell lines. These studies suggest that lowering expression of LMP-1 in EBV-associated malignancy might have therapeutic effects and might synergize with other antitumor agents. © 1998 by The American Society of Hematology.

Subcellular localization of the latent membrane protein LMP-1 in Epstein-Barr virus infected cells by immunogold EM

1994 ◽  
Vol 52 ◽  
pp. 248-249
Author(s):  
D. N. Misra ◽  
R. M. Agostini ◽  
E. J. Yunis
Keyword(s):  
Membrane Protein ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Hodgkin’S Disease ◽  
Burkitt’S Lymphoma ◽  
Latent Membrane Protein ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

Epstein-Barr virus (EBV) infection, prevalent in all human populations, is clinically silent in general, but causes infectious mononucleosis in some adolescents and B-lymphocyte proliferative disorders (LPDs) in immunocompromised individuals (e.g. AIDS infected; allograft recipients). EBV is also etiologically associated with African Burkitt’s lymphoma, nasopharyngeal carcinoma, and Hodgkin’s disease. The virus infects B lymphocytes and transforms them into lymphoblastoid cells which proliferate indefinitely in culture. The latently infected cells express an array of EBV gene products including 6 nuclear antigens (EBNAs), terminal proteins LMP-2A and LMP-2B, latent membrane protein LMP-1, and untranslated RNAs EBER 1 and EBER 2. These components are being extensively studied since they are involved in latency or proliferative transformation; LMP-1 has also shown oncogenic properties. In this work, we have used immunogold electron microscopy for precise subcellular localization of LMP-1 in EBV infected cell lines.Two human cell lines, P3HR-1 (Burkitt’s lymphoma) and CCL-113 (Hodgkin’s disease), obtained from ATCC, were grown in RPMI 1640 containing 20% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin.

The bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation

1989 ◽  
Vol 9 (2) ◽  
pp. 701-710
Author(s):  
Z Chen-Levy ◽  
J Nourse ◽  
M L Cleary
Keyword(s):  
Membrane Protein ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Quantitative Difference ◽  
Integral Membrane Protein ◽  
Lymphoid Tissues ◽  
Hydrophobic Region ◽  
Human Lymphoid Cell ◽  
Translocation Breakpoints ◽  
Lymphoid Cell Lines

We have identified a 24-kilodalton protein that is the product of the human bcl-2 gene, implicated as an oncogene because of its presence at the site of t(14;18) translocation breakpoints. The Bcl-2 protein was detected by specific, highly sensitive rabbit antibodies and was shown to be present in a number of human lymphoid cell lines and tissues, as well as in mouse B cells transfected with a bcl-2 cDNA construct. Characterization of the Bcl-2 protein demonstrated that it has a lipophilic nature and is associated with membrane structures, probably by means of its hydrophobic carboxy-terminal membrane-spanning domain. In t(14;18)-carrying cell lines, the protein is predominantly localized to the perinuclear endoplasmic reticulum, with a minor fraction in the plasma membrane. These properties, together with the observations that Bcl-2 does not have a characteristic signal peptide and is not glycosylated, suggest that it is an integral-membrane protein that spans the bilayer at its C-terminal hydrophobic region but is exposed only at the cytoplasmic surface. The relative abundance of the Bcl-2 protein in various human lymphoid cell lines correlated with transcription of the bcl-2 gene. The protein was abundant in all t(14;18)-carrying cell lines and lymphomas and was also found at lower levels in pre-B-cell lines and nonmalignant lymphoid tissues that do not carry t(14;18) translocations. These results suggest that the Bcl-2 protein is functional in normal B lymphocytes and that a quantitative difference in its expression may play a role in the pathogenesis of lymphomas carrying the t(14;18) translocation.

scholarly journals Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways

2007 ◽  
Vol 82 (4) ◽  
pp. 1946-1958 ◽  
Author(s):  
Jennifer E. Cameron ◽  
Qinyan Yin ◽  
Claire Fewell ◽  
Michelle Lacey ◽  
Jane McBride ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Latent Membrane Protein ◽  
Interferon Response ◽  
Latent Membrane Protein 1 ◽  
Type I ◽  
Induced Expression ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of nuclear factor κB (NF-κB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative reverse transcription-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-κB binding sites in the miR-146a promoter and identified a role for an Oct-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a-expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon-responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.

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