Glutathione (GSH)-decorated magnetic nanoparticles for binding glutathione-S-transferase (GST) fusion protein and manipulating live cells

2011 ◽  
Vol 2 (5) ◽  
pp. 945 ◽  
Author(s):  
Yue Pan ◽  
Marcus J. C. Long ◽  
Xinming Li ◽  
Junfeng Shi ◽  
Lizbeth Hedstrom ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Fusion Protein ◽  
Live Cells ◽  
Glutathione S Transferase ◽  
Gst Fusion Protein

scholarly journals Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

1998 ◽  
Vol 142 (4) ◽  
pp. 1063-1074 ◽  
Author(s):  
Claudio Sette ◽  
Arturo Bevilacqua ◽  
Raffaele Geremia ◽  
Pellegrino Rossi
Keyword(s):  
Fusion Protein ◽  
Tyrosine Kinase ◽  
Inositol Phosphate ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Egg Activation ◽  
Glutathione S Transferase ◽  
Truncated Form ◽  
Sh2 Domains ◽  
Gst Fusion Protein

Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit– induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267–2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the γ1 isoform of PLC (PLCγ1) competitively inhibits tr-kit– induced egg activation. A GST fusion protein containing the SH3 domain of PLCγ1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit–induced egg activation, showing that the effect of the SH3 domain of PLCγ1 is specific. Tr-kit–induced egg activation is also suppressed by co-injection of antibodies raised against the PLCγ1 SH domains, but not against the PLCγ1 COOH-terminal region. In transfected COS cells, coexpression of PLCγ1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCγ1 with respect to cells expressing PLCγ1 alone. These data indicate that tr-kit activates PLCγ1, and that the SH3 domain of PLCγ1 is essential for tr-kit–induced egg activation.

Cross-species reactivity of a monoclonal antibody against glutathione S-transferase fusion protein of human β2-adrenergic receptor

IUBMB Life ◽  
1998 ◽  
Vol 45 (2) ◽  
pp. 215-225
Author(s):  
Chan Young Shin ◽  
Suk-jo Kang ◽  
Mi-ryoung Song ◽  
Kyu Hwan Park ◽  
Dong Ook Seo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Adrenergic Receptor ◽  
Glutathione S Transferase ◽  
Β2 Adrenergic Receptor

Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger

1998 ◽  
Vol 76 (5) ◽  
pp. 837-842 ◽  
Author(s):  
Daniel Gebreselassie ◽  
Krishna Rajarathnam ◽  
Larry Fliegel
Keyword(s):  
Circular Dichroism ◽  
Membrane Protein ◽  
Fusion Protein ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Cytoplasmic Domain ◽  
Random Coil ◽  
Glutathione S Transferase ◽  
Carboxyl Terminal ◽  
Circular Dichroïsm

The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.Key words: Na+/H+ exchanger, pH regulation, membrane protein, circular dichroism.

scholarly journals Real-Time Assay of the Interaction of a GST Fusion Protein with a Protein Ligate Using Resonant Mirror Technique

BioTechniques ◽  
1997 ◽  
Vol 22 (2) ◽  
pp. 269-271 ◽  
Author(s):  
Ignacio Rubio ◽  
Philip Buckle ◽  
Hans Trutnau ◽  
Reinhard Wetzker
Keyword(s):  
Fusion Protein ◽  
Real Time ◽  
Gst Fusion Protein

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

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