• Biomolecules & Therapeutics
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• 제4권1호
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.59-59
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• 2003

Serotonin (5-HT; 5-hydroxytryptamine) exerts multiple effects on central nervous system as well as behaviors such as mood and appetite. The signaling of serotonin is mediated by 7 families of serotonin receptors, designated 5-HT$_1$ to 5-HT$_{7}$. Six families of this receptor are G-protein coupled 7TM receptors, and the third intracellular loop of these receptors is proposed to interact with specific types of G-proteins. To investigate the specific interaction between the third intracellular loop of 5-HT$_{6}$ with G$\square$s, we have constructed a chimera protein that represent the third intracellular loop of 5-HT$_{6}$ within a leucine zipper motifs, In addition an alpha subunit of human G-protein that interact with 5-HT$_{6}$ was cloned into a bacterial expression vector. The two proteins were expressed in E. coli and purified in homogeneity. The interaction of the prepared proteins was examined by ELISA assay. The affinity between the two proteins and effect of insertion mutations were discussed.ussed.d.

• Bulletin of the Korean Chemical Society
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• 제28권6호
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• pp.1005-1009
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• 2007

Leukotriene B4 (LTB4) is a potent chemoattractant for leukocytes and considered to be an inflammatory mediator. Human BLT2 (hBLT2) is a low-affinity G-protein coupled receptor for LTB4 and mediates pertussis toxin-sensitive chemotactic cell movement. Here, we dissected the interaction between hBLT2 and G-protein alpha subunits using GST fusion proteins containing intracellular regions of hBLT2 and various Gα protein including Gα i1, Gα i2, Gα i3, Gα s1, Gα o1, and Gα z. Among the tested Gα subunits, Gα i3 showed the highest binding to the third intracellular loop region of hBLT2 with a dissociation constant (KD) of 5.0 × 10?6 M. These results suggest that Gα i3 has the highest affinity to hBLT2, and the third intracellular loop region of hBLT2 is the major component for the interaction with Gα i3.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.93-93
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• 1997

Analysis of protein is often frustrated by the inability to isolate large amounts of purified protein from a native source. To overcome this problem, fusion protein expression systems such as pGEX system have been widely used. Using pGEX system, the desired protein could be easily obtained in a large amount in E. coli, and then the fusion protein could be used for the study of the function of the given protein. To analyze and purify the GST fusion protein, anti-GST antibody could be used as one of the system of choice. However, the production and characterization of monoclonal anti-GST antibody has not been studied extensively yet. To produce monoclonal anti-GST antibody, GST was purified from E. coli transformed with pGEX-cs, one of the pGEX system and was used as an antigen. The monoclonal antibody was produced by fusion of the immunized spleen cells with SP2-0 myeloma cells. The antibody was characterized by ELISA, western blotting, etc. The monoclonal antibody produced in this study (mAb-GSTA) showed strong and specific immunoreactivity against not only GST but also GST-fusion proteins. Also, mAb-GSTA was successfully used for the immunoaffinity purification of the GST ${\beta}$-Rc.-third intracellular-loop fusion protein. The results of the present study suggest that mAb-GSTA may be used for the identification and purification of GST fusion proteins.