Production of Monoclonal Antibody Against Human 14 - 3 - 3 Zeta Isoform Expressed in Escherichia coli.
- Author:
Byung Uk LIM
;
Jae Seung KANG
;
Jung Woo KIM
;
Nam Jong CHOI
- Publication Type:Original Article
- Keywords:
Monoclonal antibody;
Expression
- MeSH:
14-3-3 Proteins;
Animals;
Antibodies, Monoclonal;
Cell Line;
Chromatography, Affinity;
Clone Cells;
Cytoplasm;
DNA, Complementary;
Escherichia coli*;
Escherichia*;
HeLa Cells;
Histidine;
Humans*;
Hybridomas;
Immune Sera;
Mice;
Nickel;
Phosphoserine;
Protein Isoforms;
Signal Transduction;
Staphylococcal Protein A
- From:Korean Journal of Immunology
1997;19(3):391-398
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
14-3-3 proteins are cytoplasmic proteins of about 29 kDa and have a minimum of seven isoforms. This protein is important in signal transduction with the ability of binding with phosphoserine of many signalling proteins. We expressed 14-3-3 protein tagged with 6 histidine residues in E. coli and purified the protein by nickel affinity chromatography. Using this purified protein as an antigen, we made rabbit antisera and mouse monoclonal antibodies to 14-3-3 zeta isoform. We subcloned cDNA of 14-3-3 zeta isoform derived from HeLa cell lamda gt 11 library into an E. coli expression vector which is designed to express heterologous protein with N- terminal 6 hidtidine tag. BALB/c mice were immunized with purified 14-3-3 protein and the hybridoma clones which produce monoclonal antibodies angainst 14-3-3 protein were selected. These monoclonal antibodies reacted with the recombinant protein expressed in E. coli as well as the 29-kDa native protein in various cell lines. However, they did not immunoprecipitate 14-3-3 protein. The monoclonal antibodies produced in this study can be valuable tools for the identification of the 14-3-3 in signal transduction study.
