scholarly journals Codon Optimization of Insect Odorant Receptor Genes May Increase Their Stable Expression for Functional Characterization in HEK293 Cells

2021 ◽  
Vol 15 ◽  
Author(s):  
Rebecca E. Roberts ◽  
Jothi Kumar Yuvaraj ◽  
Martin N. Andersson
Keyword(s):  
Bark Beetles ◽  
Codon Optimization ◽  
Odorant Receptor ◽  
Functional Characterization ◽  
Stable Expression ◽  
Hek293 Cells ◽  
Western Blots ◽  
Protein Levels ◽  
Hek Cells

Insect odorant receptor (OR) genes are routinely expressed in Human Embryonic Kidney (HEK) 293 cells for functional characterization (“de-orphanization”) using transient or stable expression. However, progress in this research field has been hampered because some insect ORs are not functional in this system, which may be due to insufficient protein levels. We investigated whether codon optimization of insect OR sequences for expression in human cells could facilitate their functional characterization in HEK293 cells with stable and inducible expression. We tested the olfactory receptor co-receptor (Orco) proteins from the bark beetles Ips typographus (“Ityp”) and Dendroctonus ponderosae (“Dpon”), and six ItypORs previously characterized in Xenopus laevis oocytes and/or HEK cells. Western blot analysis indicated that codon optimization yielded increased cellular protein levels for seven of the eight receptors. Our experimental assays demonstrated that codon optimization enabled functional characterization of two ORs (ItypOR25 and ItypOR29) which are unresponsive when expressed from wildtype (non-codon optimized) genes. Similar to previous Xenopus oocyte recordings, ItypOR25 responded primarily to the host/conifer monoterpene (+)-3-carene. ItypOR29 responded primarily to (+)-isopinochamphone and similar ketones produced by fungal symbionts and trees. Codon optimization also resulted in significantly increased responses in ItypOR49 to its pheromone ligand (R)-(−)-ipsdienol, and improved responses to the Orco agonist VUAA1 in ItypOrco. However, codon optimization did not result in functional expression of DponOrco, ItypOR23, ItypOR27, and ItypOR28 despite higher protein levels as indicated by Western blots. We conclude that codon optimization may enable or improve the functional characterization of insect ORs in HEK cells, although this method is not sufficient for all ORs that are not functionally expressed from wildtype genes.

Urine Proteomics Profiling and Functional Characterization of Knee Osteoarthritis Using iTRAQ Technology

2019 ◽  
Vol 51 (11) ◽  
pp. 735-740 ◽  
Author(s):  
Ke Xiao ◽  
Lingjia Yu ◽  
Lisi Zhu ◽  
Zhihong Wu ◽  
Xisheng Weng ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Joint Function ◽  
Urine Protein ◽  
Western Blots ◽  
Urine Proteomics ◽  
Protein Levels ◽  
Mass Spectrometry Technology ◽  
Proteome Profile ◽  
Insight Into

AbstractOsteoarthritis (OA) is a degenerative chronic disease affecting the whole joint structures. With the increment in life expectancy and aging population, OA has become one of the largest socioeconomic burdens, associated with pain and loss of joint function. However, early laboratory tests of OA are still lacking. Therefore, new diagnostic tests for this disease are urgently needed. In this study, to gain an insight into the pathogenesis and the potential biomarkers of OA, we implemented a comparative urine proteomics study on OA patients and health people using iTRAQ-based mass spectrometry technology. Western blotting was used to validate the relative changes in urine protein levels for four of the identified proteins. We constructed a comprehensive urine proteome profile of the OA patients and identified 102 proteins differently changed in abundance. Forty-six proteins were upregulated and 56 proteins were significantly downregulated in OA patients. Furthermore, the proteins, COL-4, MMP9, adiponectin, and BBOX1 were validated through Western blots, which can serve as valuable candidate biomarkers and help to illustrate the pathogenesis of OA. These findings may provide clues for promising biomarkers for the early diagnosis and also offer a theoretical basis for the early treatment of OA.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

scholarly journals Characterization of an N-terminal Nav1.5 channel variant – a potential risk factor for arrhythmias and sudden death?

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Stefanie Scheiper-Welling ◽  
Paolo Zuccolini ◽  
Oliver Rauh ◽  
Britt-Maria Beckmann ◽  
Christof Geisen ◽  
...  
Keyword(s):  
Sudden Death ◽  
Functional Characterization ◽  
Missense Variant ◽  
Hek293 Cells ◽  
Patch Clamp Technique ◽  
Gene Encoding ◽  
Heterozygous Variant ◽  
Cardiac Sodium Channel ◽  
Electrophysiological Measurements

Abstract Background Alterations in the SCN5A gene encoding the cardiac sodium channel Nav1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. Methods Mutant as well as wild type GFP tagged Nav1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. Results Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Nav1.5 current over the entire voltage window. Conclusion The results support the assumption that the detected sequence aberration alters Nav1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death.

Identification and functional characterization of an odorant receptor in pea aphid, Acyrthosiphon pisum

2017 ◽  
Vol 26 (1) ◽  
pp. 58-67 ◽  
Author(s):  
Rui-Bin Zhang ◽  
Yang Liu ◽  
Shan-Chun Yan ◽  
Gui-Rong Wang
Keyword(s):  
Acyrthosiphon Pisum ◽  
Odorant Receptor ◽  
Functional Characterization ◽  
Pea Aphid

scholarly journals Characterization of Na+/H+ exchanger NHE8 in cultured renal epithelial cells

2007 ◽  
Vol 293 (3) ◽  
pp. F761-F766 ◽  
Author(s):  
Jianning Zhang ◽  
Ion Alexandru Bobulescu ◽  
Sunita Goyal ◽  
Peter S. Aronson ◽  
Michel G. Baum ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Protein Levels ◽  
Primary Amino ◽  
Interfering Rna

NHE8 is expressed in the apical membrane of the proximal tubule and is predicted to be a Na+/H+ exchanger on the basis of its primary amino acid sequence. Functional characterization of native NHE8 in mammalian cells has not been possible to date. We screened a number of polarized renal cell lines for the plasma membrane Na+/H+ exchangers (NHE1, 2, 3, 4, and 8) and found only NHE1 and NHE8 transcripts in NRK cells by RT-PCR. NHE8 protein is expressed in the apical membrane of NRK cells as demonstrated by immunoblots, confocal fluorescent immunocytochemistry, and immunoelectron microscopy. NHE1, on the other hand, is expressed primarily in the basolateral membrane. Bilateral perfusion of NRK cells grown on permeable supports shows Na+/H+ exchange activity on both the apical and basolateral membranes. NHE8-specific small interfering RNA knocks down NHE8 protein expression but does not affect NHE1 protein levels. Knockdown of NHE8 protein is accompanied by a commensurate reduction in apical NHE activity, without altered basolateral NHE activity. Conversely, transfection of NHE1-specific small interfering RNA knocks down NHE1 protein expression without affecting NHE8 protein levels and reduces basolateral NHE activity without affecting apical NHE activity. NHE8 is the only apical membrane Na+/H+ exchanger in NRK cells. NHE8 activity is Na+ dependent, displaying a cooperative sigmoidal relationship, and is highly sensitive to 5-( N-ethyl- n-isopropyl)-amiloride (EIPA). NRK cells provide a useful system where NHE8 can be studied in its native environment.

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