scholarly journals Development and Characterization of Monoclonal Antibodies Specific to the Serotonin 5-HT2A Receptor

1998 ◽  
Vol 46 (7) ◽  
pp. 811-824 ◽  
Author(s):  
Chun Wu ◽  
Elizabeth J. Yoder ◽  
Jean Shih ◽  
Kevin Chen ◽  
Peter Dias ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Rat Brain ◽  
Pyramidal Neurons ◽  
Expression System ◽  
Molecular Size ◽  
Baculovirus Expression System ◽  
Differential Staining ◽  
Receptor Subtypes ◽  
And Function

Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT2A receptor. To generate MAbs against 5-HT2AR, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST-5-HT2AR recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST-5-HT2AR, rat and human brain lysate, and lysate from cell lines transfected with 5-HT2AR cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT2AR N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT2AR in lysates from rat brain and a 5-HT2AR-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT2AR but not with recombinant 5-HT2BR and 5-HT2CR. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies.

Characterization of glycoengineered anti-HER2 monoclonal antibodies produced by using a silkworm–baculovirus expression system

2018 ◽  
Vol 163 (6) ◽  
pp. 481-488 ◽  
Author(s):  
Yuriko Egashira ◽  
Satoru Nagatoishi ◽  
Masato Kiyoshi ◽  
Akiko Ishii-Watabe ◽  
Kouhei Tsumoto
Keyword(s):  
Monoclonal Antibodies ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Baculovirus Expression

Expression, Purification, and Characterization of Recombinant Human α1-Antitrypsin Produced Using Silkworm–Baculovirus Expression System

2018 ◽  
Vol 60 (12) ◽  
pp. 924-934 ◽  
Author(s):  
Yoshiki Morifuji ◽  
Jian Xu ◽  
Noriko Karasaki ◽  
Kazuhiro Iiyama ◽  
Daisuke Morokuma ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Purification And Characterization ◽  
Baculovirus Expression

scholarly journals Central distribution and function of 5-HTe and 5-HT7 receptor subtypes in the rat brain

1997 ◽  
Vol 73 ◽  
pp. 13
Author(s):  
Mitsuhiro Yoshioka ◽  
Machiko Matsumoto ◽  
Hiroko Togashi ◽  
Kjyoshi Mori ◽  
Hideya Saito
Keyword(s):  
Rat Brain ◽  
Receptor Subtypes ◽  
And Function

scholarly journals Characterization and Reconstitution of Drosophila γ-Tubulin Ring Complex Subunits

2000 ◽  
Vol 151 (7) ◽  
pp. 1513-1524 ◽  
Author(s):  
Ruwanthi N. Gunawardane ◽  
Ona C. Martin ◽  
Kan Cao ◽  
Lijun Zhang ◽  
Kimberly Dej ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Sequence Motifs ◽  
Microtubule Nucleation ◽  
Sequence Comparisons ◽  
Baculovirus Expression ◽  
Ring Complex ◽  
And Function ◽  
First Time ◽  
Centrosomal Proteins

The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin, the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC, we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.

scholarly journals Partial characterization of natural and recombinant human soluble CD23

1992 ◽  
Vol 286 (3) ◽  
pp. 819-824 ◽  
Author(s):  
K Rose ◽  
G Turcatti ◽  
P Graber ◽  
S Pochon ◽  
P O Regamey ◽  
...  
Keyword(s):  
Insect Cells ◽  
Peptide Mapping ◽  
Sequence Similarity ◽  
Expression System ◽  
Protein A ◽  
Baculovirus Expression System ◽  
Partial Characterization ◽  
Disulphide Bonds ◽  
Baculovirus Expression

The purification to homogeneity of an active soluble 25 kDa fragment of CD23, produced in insect cells using the baculovirus expression system, is described. Peptide mapping and analysis by Edman degradation and mass spectrometry permitted partial characterization of the protein. A total of 165 out of 172 residues, including N-terminal and C-terminal regions, were mapped. The positions of the two disulphide bonds in the IgE-binding region were also determined: residue 110 is joined to residue 124, and residue 42 to residue 133. Natural CD23 25 kDa fragment was also analysed and found to possess the same disulphide bond arrangement. These results extend the previously noted sequence similarity with lectins to elements of secondary structure.

scholarly journals Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV

2006 ◽  
Vol 396 (2) ◽  
pp. 391-399 ◽  
Author(s):  
Jais R. Bjelke ◽  
Jesper Christensen ◽  
Per F. Nielsen ◽  
Sven Branner ◽  
Anders B. Kanstrup ◽  
...  
Keyword(s):  
Peptide Yy ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Dipeptidyl Peptidases ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Dipeptidyl Peptidase Iv Inhibition

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis–Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar Ki values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.

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