scholarly journals Cell Surface-Exposed Tetanus Toxin Fragment C Produced by Recombinant Bacillus anthracis Protects against Tetanus Toxin

1999 ◽  
Vol 67 (9) ◽  
pp. 4847-4850 ◽  
Author(s):  
Stéphane Mesnage ◽  
Martine Weber-Levy ◽  
Michel Haustant ◽  
Michèle Mock ◽  
Agnès Fouet
Keyword(s):  
Cell Surface ◽  
Bacillus Anthracis ◽  
Tetanus Toxin ◽  
Surface Display ◽  
Humoral Response ◽  
Cell Surface Display ◽  
Hybrid Protein ◽  
A Cell ◽  
Component Gene

Bacillus anthracis, the causal agent of anthrax, synthesizes two surface layer (S-layer) proteins, EA1 and Sap, which account for 5 to 10% of total protein and are expressed in vivo. A recombinant B. anthracis strain was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and tetanus toxin fragment C (ToxC). This construct was expressed under the control of the promoter of the S-layer component gene. The hybrid protein was stably expressed on the cell surface of the bacterium. Mice were immunized with bacilli of the corresponding strain, and the hybrid protein elicited a humoral response to ToxC. This immune response was sufficient to protect mice against tetanus toxin challenge. Thus, the strategy developed in this study may make it possible to generate multivalent live veterinary vaccines, using the S-layer protein genes as a cell surface display system.

scholarly journals Development of a cell surface display system in Chlamydomonas reinhardtii

2022 ◽  
Vol 61 ◽  
pp. 102570
Author(s):  
João Vitor Dutra Molino ◽  
Roberta Carpine ◽  
Karl Gademann ◽  
Stephen Mayfield ◽  
Simon Sieber
Keyword(s):  
Cell Surface ◽  
Chlamydomonas Reinhardtii ◽  
Surface Display ◽  
Cell Surface Display ◽  
Display System ◽  
A Cell ◽  
Cell Surface Display System

scholarly journals Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response

2006 ◽  
Vol 72 (11) ◽  
pp. 7140-7147 ◽  
Author(s):  
Frank Breinig ◽  
Björn Diehl ◽  
Sabrina Rau ◽  
Christian Zimmer ◽  
Helmut Schwab ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
Constitutive Activation ◽  
Unfolded Protein ◽  
Yeast Cell Surface ◽  
Protein Response

ABSTRACT Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

scholarly journals Rapidly rendering cells phagocytic through a cell surface display technique and concurrent Rac activation

2014 ◽  
Vol 7 (334) ◽  
pp. rs4-rs4 ◽  
Author(s):  
H. Onuma ◽  
T. Komatsu ◽  
M. Arita ◽  
K. Hanaoka ◽  
T. Ueno ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
A Cell ◽  
Display Technique

scholarly journals A cell surface display fluorescent biosensor for measuring MMP14 activity in real-time

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Alexander Braun ◽  
Matthew J. Farber ◽  
Zachary A. Klase ◽  
Peter B. Berget ◽  
Kenneth A. Myers
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Surface Display ◽  
Cell Surface Display ◽  
Fluorescent Biosensor ◽  
A Cell

scholarly journals A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.

1994 ◽  
Vol 126 (4) ◽  
pp. 979-989 ◽  
Author(s):  
S Corvera ◽  
A Chawla ◽  
R Chakrabarti ◽  
M Joly ◽  
J Buxton ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Surface Display ◽  
Cell Surface Display ◽  
Underlying Mechanism ◽  
Terminal Domain ◽  
Amino Acid Region ◽  
Intracellular Compartments ◽  
A Cell ◽  
Glucose Transporter Glut4

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 degrees resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transferrin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady-state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.

scholarly journals Development of a yeast cell surface display method using the SpyTag/SpyCatcher system

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaho Kajiwara ◽  
Wataru Aoki ◽  
Naoki Koike ◽  
Mitsuyoshi Ueda
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Yeast Cell ◽  
Directed Evolution ◽  
Surface Display ◽  
Cell Surface Display ◽  
Protein Ligation ◽  
Continuous Evolution ◽  
Yeast Cell Surface

AbstractYeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.

scholarly journals Development of a Cell Surface Display System in a Magnetotactic Bacterium, “Magnetospirillum magneticum” AMB-1

2008 ◽  
Vol 74 (11) ◽  
pp. 3342-3348 ◽  
Author(s):  
Masayoshi Tanaka ◽  
Yuko Nakata ◽  
Tetsushi Mori ◽  
Yoshiko Okamura ◽  
Hitoshi Miyasaka ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Cell Surface ◽  
Magnetic Force ◽  
Secondary Structure Prediction ◽  
Surface Display ◽  
Cell Surface Display ◽  
Display System ◽  
A Cell ◽  
Magnetospirillum Magneticum ◽  
Cell Surface Display System

ABSTRACT Bacterial cell surface display is a widely used technology for bioadsorption and for the development of a variety of screening systems. Magnetotactic bacteria are unique species of bacteria due to the presence of magnetic nanoparticles within them. These intracellular, nanosized (50 to 100 nm) magnetic nanoparticles enable the cells to migrate and be manipulated by magnetic force. In this work, using this unique characteristic and based on whole-genomic and comprehensive proteomic analyses of these bacteria, a cell surface display system has been developed by expressing hexahistidine residues within the outer coiled loop of the membrane-specific protein (Msp1) of the “Magnetospirillum magneticum” (proposed name) AMB-1 bacterium. The optimal display site of the hexahistidine residues was successfully identified via secondary structure prediction, immunofluorescence microscopy, and heavy metal binding assay. The established AMB-1 transformant showed high immunofluorescence response, high Cd2+ binding, and high recovery efficiency in comparison to those of the negative control when manipulated by magnetic force.

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