Expression system for enhanced green fluorescence protein conjugated recombinant antibody fragment.
ABSTRACT Recent development of recombinant antibody technology has enabled fusion of recombinant antibody fragment with fluorescent proteins for various applications such as flow cytometry, fluorescence immunoassay, and fluorescent microscopy. In this study, we generated various forms of green fluorescence protein (EGFP)-fused anti-c-Met antibody fragment. Among these fusion proteins, EGFP fusion to the light chain showed high expression in a soluble form of protein in E. coli, and high binding activity to c-Met. A feasibility of the constructs was further examined by replacing the Fab gene by a Fab library of catalytic subunit of protein kinase A (PKA) to construct the Fab library in EGFP fused form. We also constructed the conventional Fab library. After a series of biopanning, we found that the binding capability of EGFP-anti-PKA Fab was comparable with anti-PKA Fab. Sequence analysis of the selected clones showed > or =99% identity in amino acid sequence and shared the same CDR sequence. These results demonstrate that EGFP fusion to the light chain using our vector system does not influence the selection of reactive Fab and that this vector system is useful for EGFP fusion to Fab to develop a one-step detection system.
- Methods in Enzymology 02/1983; 99:51-5. · 2.00 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Transcription factors (TFs) have been difficult to identify by bioinformatics, due to the heterogenous nature of the domains they are composed of. Therefore, we have developed a simple and generally applicable screening system for the identification of transcriptional activators, based on the presence of a functional transactivation domain (TAD). The system utilizes a retroviral vector to express a cDNA library as fusion genes with the yeast gal4 DNA-binding domain. This retroviral library is transduced into a murine NIH3T3-based reporter cell line carrying a stable integrated gal4 promoter-green fluorescent protein reporter gene. cDNA inserts encoding a functional TAD reconstitute a chimeric TF that activates the reporter gene. After fluorescence activated cell sorting (FACS) and expansion of GFP-positive cells, the responsible cDNA inserts are retrieved. From a cDNA library of cytokine-stimulated human umbilical vein endothelial cells (HUVEC), a number of known as well as potentially novel TFs were isolated, demonstrating the suitability of the system. The identification of other factors that are currently not associated with transcriptional regulation suggest additional functions for these proteins. Moreover, our results have focused attention on signaling pathways that have not been recognized previously in the context of endothelial cell biology.Nucleic Acids Research 09/2002; 30(16):e80. · 8.28 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The family of insulin receptor substrates (IRSs) has been reported to play important roles for signal transduction of various hormones. Four members of the IRS family have been described. Each IRS is believed to have different functions; however, the distinct physiological roles of each IRS are unclear. This study was undertaken to determine the intracellular localization of IRS-3. IRS-3 was expressed in COS-7 cells as fusion with a green fluorescent protein (GFP), and subcellular localization of the chimera protein was analyzed by fluorescent microscopy. Surprisingly, GFP-IRS-3 was localized not only adjacent to the plasma membrane but also in the nucleus. We confirmed by immunostaining with anti-IRS-3 antibody that non-fused IRS-3 protein is also localized in the nucleus of COS-7 cells that were transfected with IRS-3 cDNA. In addition, we detected endogenous IRS-3 in the nucleus of isolated rat adipocytes. We then studied subcellular localization of deletion mutants and fragments of IRS-3 fused with GFP. We found that the region corresponding to amino acid residues 192-223 in the phosphotyrosine binding domain played an important role in nuclear localization. This region includes sequences that are unique to IRS-3. We then investigated intracellular localization of other IRSs fused with GFP. GFP-IRS-1, GFP-IRS-2, and GFP-IRS-4 were mainly localized in the cytosol or plasma membranes. Chimeric protein, Gal4 DNA binding domain fused with IRS-3 C-terminal region, increased transcription of the reporter gene containing Gal4 binding site in human embryonic kidney 293 cells. These results suggest that intracellular localization of IRS-3 is determined by a different mechanism from other IRS proteins, and that IRS-3 possesses a transcription-regulating activity.Journal of Biological Chemistry 04/2002; 277(9):6846-51. · 4.65 Impact Factor