scholarly journals The Collagen V Homotrimer[α1(V)]3Production Is Unexpectedly Favored over the Heterotrimer[α1(V)]2α2(V)in Recombinant Expression Systems

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Muriel Roulet ◽  
Merja Välkkilä ◽  
Hélène Chanut-Delalande ◽  
Eija-Riitta Hämäläinen ◽  
Efrat Kessler ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Molecular Form ◽  
Recombinant Expression ◽  
Expression Systems ◽  
Key Factors ◽  
Double Immunofluorescence ◽  
Hek 293 ◽  
Transfected Cells ◽  
Biomedical Device ◽  
Cell Expression

Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer[α1(V)]2α2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proα1(V) and proα2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of[α1(V)]3homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proα2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection withHsp47cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen Vα-chains showed that, contrary to fibroblasts, collagen Vα-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly.

scholarly journals Properties of WT and mutant hERG K+ channels expressed in neonatal mouse cardiomyocytes

2010 ◽  
Vol 298 (6) ◽  
pp. H1842-H1849 ◽  
Author(s):  
Eric C. Lin ◽  
Katherine M. Holzem ◽  
Blake D. Anson ◽  
Brooke M. Moungey ◽  
Sadguna Y. Balijepalli ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Neonatal Mouse ◽  
Delayed Rectifier ◽  
Missense Mutations ◽  
Expression Systems ◽  
Hek 293 ◽  
Deactivation Kinetics ◽  
Pharmacological Correction ◽  
Cell Expression ◽  
Herg Channels

Mutations in human ether-a-go-go-related gene 1 ( hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming α-subunits that coassemble to form rapidly activating delayed rectifier K+ current in the heart. LQT2-linked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-linked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-linked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The ΔY475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue.

Physiologic Regulation of Systemic Klotho Levels by Renal CaSR Signaling in Response to CaSR Ligands and pHo

2021 ◽  
pp. ASN.2021020276
Author(s):  
Joonho Yoon ◽  
Zhenan Liu ◽  
Eunyoung Lee ◽  
Liping Liu ◽  
Silvia Ferre ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Expression Systems ◽  
Ckd Progression ◽  
Hek 293 ◽  
Lateral Membrane ◽  
Physiologic Mechanism ◽  
293 Cells ◽  
Alkali Therapy ◽  
Cell Expression ◽  
Physiologic Regulation

Background: The kidney is the source of sKlotho and kidney-specific loss of Klotho leads to a phenotype resembling the premature multi-organ failure phenotype in Klothohypomorphic mice (kl/kl mice). Klotho and the Ca-sensing receptor (CaSR) are highly expressed in the distal convoluted tubule (DCT). The physiologic mechanisms that regulate sKlotho levels are unknown. Methods: We measured sKlotho in WT and tubule-specific CaSR-/- (TS-CaSR-/-) mice treated with calcimimetics, alkali, or acid, and Klotho shed from minced mouse kidneys, as well as from HEK-293 cells expressing the CaSR and Klotho, in response to calcimimetics, calcilytics, alkalotic and acidic pH, and ADAM protease inhibitors. The CaSR, Klotho, and ADAM10 were imaged in mouse kidneys and cell expression systems using confocal microscopy. Results: The CaSR, Klotho, and ADAM10 co-localize on the baso-lateral membrane of the DCT. Calcimimetics and HCO3 increase serum sKlotho levels in WT but not in CaSR-/- mice and acid pH suppresses sKlotho levels in WT mice. In minced kidneys and cultured cells, CaSR activation with high Ca, calcimimetics, or alkali increase shed Klotho levels via ADAM10, as demonstrated using the ADAM10 inhibitor GI254023X and siRNA. In cultured cells the CaSR, Klotho, and ADAM10 form cell surface aggregates that disperse following CaSR activation. Conclusions: We identify a novel physiologic mechanism for regulation of sKlotho levels by the renal CaSR-ADAM10-Klotho pathway. We show that CaSR activators, including alkali, increase renal CaSR-stimulated Klotho shedding and predict that this mechanism is relevant to the effects of acidosis and alkali therapy on CKD progression.

scholarly journals Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines

2005 ◽  
Vol 389 (1) ◽  
pp. 241-247 ◽  
Author(s):  
Yasuo OKAMOTO ◽  
Jun MORISHITA ◽  
Jun WANG ◽  
Patricia C. SCHMID ◽  
Randy J. KREBSBACH ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Fold Increase ◽  
Living Cells ◽  
Animal Tissues ◽  
Hek 293 ◽  
Transfected Cells ◽  
Mammalian Cell Lines

In animal tissues, NAEs (N-acylethanolamines), including N-arachidonoylethanolamine (anandamide), are primarily formed from their corresponding NAPEs (N-acylphosphatidylethanolamines) by a phosphodiesterase of the PLD (phospholipase D) type (NAPE-PLD). Recently, we cloned cDNAs of NAPE-PLD from mouse, rat and human [Okamoto, Morishita, Tsuboi, Tonai and Ueda (2004) J. Biol. Chem. 279, 5298–5305]. However, it remained unclear whether NAPE-PLD acts on endogenous NAPEs contained in the membrane of living cells. To address this question, we stably transfected two mammalian cell lines (HEK-293 and CHO-K1) with mouse NAPE-PLD cDNA, and investigated the endogenous levels and compositions of NAPEs and NAEs in these cells, compared with mock-transfected cells, with the aid of GC-MS. The overexpression of NAPE-PLD caused a decrease in the total amount of NAPEs by 50–90% with a 1.5-fold increase in the total amount of NAEs, suggesting that the recombinant NAPE-PLD utilizes endogenous NAPE as a substrate in the cell. Since the compositions of NAEs and NAPEs of NAPE-PLD-overexpressing cells and mock-transfected cells were very similar, the enzyme did not appear to discriminate among the N-acyl groups of endogenous NAPEs. These results confirm that overexpressed NAPE-PLD is capable of forming NAEs, including anandamide, in living cells.

scholarly journals Recombinant expression of monovalent and bivalent anti-TNT-antibodies: Evaluation of different expression systems

2008 ◽  
Vol 73 (2) ◽  
pp. 139-145 ◽  
Author(s):  
Mladen Simonovic ◽  
Svetlana Zlatanovic-Milosevic ◽  
Miroslav Vrvic ◽  
Branislav Simonovic
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Recombinant Expression ◽  
Methylotrophic Yeast ◽  
Eukaryotic Expression ◽  
Expression Systems ◽  
Yeast Pichia Pastoris ◽  
Expression In Escherichia Coli ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Eukaryotic Expression Systems

Monoclonal 11B3 anti-TNT (trinitrotoluene) antibody was expressed as a monovalent and bivalent form using different prokaryotic and eukaryotic expression systems. Recombinant expression in Escherichia coli, mammalian cells and the methylotrophic yeast Pichia pastoris was performed to obtain disulfide-linked and glycosylated antibody forms. The generation of antibody and subsequent evaluation of the expression rates were performed using intracellular, excretory and periplasmatic expression techniques. All methods involved striving for native expressed antibody with maintenance of its functionality only.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

scholarly journals Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens

2002 ◽  
Vol 70 (3) ◽  
pp. 1121-1128 ◽  
Author(s):  
Kent B. Marty ◽  
Christopher L. Williams ◽  
Linda J. Guynn ◽  
Michael J. Benedik ◽  
Steven R. Blanke
Keyword(s):  
Cytotoxic Activity ◽  
Serratia Marcescens ◽  
Mammalian Cells ◽  
Divalent Cations ◽  
Recombinant Expression ◽  
Cytotoxic Factor ◽  
Culture Filtrates ◽  
Type Strains

ABSTRACT Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.

scholarly journals The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins

2021 ◽  
pp. 153537022110301
Author(s):  
Caio Coutinho de Souza ◽  
Jander Matos Guimarães ◽  
Soraya dos Santos Pereira ◽  
Luis André Morais Mariúba
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Recombinant Expression ◽  
Academic Research ◽  
Future Research ◽  
Expression Systems ◽  
Bioactive Substances ◽  
Exogenous Dna ◽  
Chemical Inducers

Bacillus subtilis is a successful host for producing recombinant proteins. Its GRAS (generally recognized as safe) status and its remarkable innate ability to absorb and incorporate exogenous DNA into its genome make this organism an ideal platform for the heterologous expression of bioactive substances. The factors that corroborate its value can be attributed to the scientific knowledge obtained from decades of study regarding its biology that has fostered the development of several genetic engineering strategies, such as the use of different plasmids, engineering of constitutive or double promoters, chemical inducers, systems of self-inducing expression with or without a secretion system that uses a signal peptide, and so on. Tools that enrich the technological arsenal of this expression platform improve the efficiency and reduce the costs of production of proteins of biotechnological importance. Therefore, this review aims to highlight the major advances involving recombinant expression systems developed in B. subtilis, thus sustaining the generation of knowledge and its application in future research. It was verified that this bacterium is a model in constant demand and studies of the expression of recombinant proteins on a large scale are increasing in number. As such, it represents a powerful bacterial host for academic research and industrial purposes.

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