Comparison of mutant forms of the green fluorescent protein as expression markers in Chinese hamster ovary (CHO) and Saccharomyces cerevisiae cells

1998 ◽  
Vol 62 (1) ◽  
pp. 29-45 ◽  
Author(s):  
A Natarajan ◽  
S Subramanian ◽  
F Srienc
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Green Fluorescent ◽  
Mutant Forms

scholarly journals The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

2003 ◽  
Vol 372 (2) ◽  
pp. 335-345 ◽  
Author(s):  
Martin E. LIDELL ◽  
Malin E. V. JOHANSSON ◽  
Matthias MÖRGELIN ◽  
Noomi ASKER ◽  
James R. GUM ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Full Length ◽  
Globular Domain ◽  
Wild Type ◽  
C Terminus ◽  
Green Fluorescent

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin κ-chain signal sequence to direct the protein to the secretory pathway. Pulse–chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.

Hands-on experiments on glycemia regulation and type 1 diabetes

2015 ◽  
Vol 39 (3) ◽  
pp. 232-239 ◽  
Author(s):  
M. Mingueneau ◽  
A. Chaix ◽  
N. Scotti ◽  
J. Chaix ◽  
A. Reynders ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pancreatic Cells ◽  
Green Fluorescent

In the present article, we describe a 3-day experimental workshop on glycemia regulation and type 1 diabetes that engages students in open-ended investigations and guided experiments leading to results that are not already known to them. After an initial questioning phase during which students observe PowerPoint slides depicting the glycemia (blood glucose levels) of individuals in various situations, students design, execute, and interpret experiments to address one of the following questions: 1) Which criteria must an animal model of diabetes fulfill? 2) How do pancreatic cells maintain glycemia constant? and 3) Is there a way to produce an insulin protein similar to the one released by human pancreatic cells? Students then 1) measure glycemia and glycosuria in control mice and in a mouse model of type 1 diabetes (Alloxan-treated mice), 2) measure the release of insulin by pancreatic β-cells (INS-1 cell line) in response to different concentrations of glucose in the extracellular medium, and 3) transfect Chinese hamster ovary cells with a plasmid coding for green fluorescent protein, observe green fluorescent protein fluorescence of some of the transfected Chinese hamster ovary cells under the microscope, and observe the characteristics of human insulin protein and its three-dimensional conformation using RASMOL software. At the end of the experimental session, students make posters and present their work to researchers. Back at school, they may also present their work to their colleagues.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

2004 ◽  
Vol 70 (2) ◽  
pp. 961-966 ◽  
Author(s):  
Antje Eiden-Plach ◽  
Tatjana Zagorc ◽  
Tanja Heintel ◽  
Yvonne Carius ◽  
Frank Breinig ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Schizosaccharomyces Pombe ◽  
Protein Secretion ◽  
Candida Glabrata ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Foreign Protein ◽  
Green Fluorescent

ABSTRACT Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

Green fluorescent protein (GFP) tagged to the cytoplasmic tail of αIIb or β3 allows the expression of a fully functional integrin αIIbβ3: effect of β3GFP on αIIbβ3 ligand binding

2001 ◽  
Vol 357 (2) ◽  
pp. 529-536 ◽  
Author(s):  
Sébastien PLANÇON ◽  
Marie-Christine MOREL-KOPP ◽  
Elisabeth SCHAFFNER-RECKINGER ◽  
Ping CHEN ◽  
Nelly KIEFFER
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Cytoplasmic Tail ◽  
Receptor Activation ◽  
Living Cells ◽  
Cell Surface Expression ◽  
Β3 Integrin ◽  
Green Fluorescent

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a β3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either αIIb or β3 allowed normal expression, heterodimerization, processing and surface exposure of αIIbGFPβ3 and αIIbβ3GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced αIIbβ3 capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged αIIbβ3. GFP-tagged αIIbβ3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125FAK tyrosine phosphorylation similar to wild-type αIIbβ3 (where FAK corresponds to focal adhesion kinase). However, GFP tagged to β3, but not to αIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of αIIbβ3 receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged αIIbβ3 during the early stages of cell attachment and spreading, starting with αIIbβ3 clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of αIIbβ3 in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either αIIb or β3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the β3 integrin cytoplasmic tail, rather than the αIIb subunit, plays a major role in αIIbβ3 affinity modulation. With the successful direct visualization of functional αIIbβ3 receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

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