Article

Expression system for enhanced green fluorescence protein conjugated recombinant antibody fragment.

Department of Biochemistry and Institute of Cardiovascular Research, Chonbuk National University Medical School, Chonju, Korea.
Hybridoma and Hybridomics 11/2004; 23(5):279-86. DOI: 10.1089/hyb.2004.23.279
Source: PubMed

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.

0 Bookmarks
 · 
87 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: According to World Health Organization (WHO), cancer is a leading cause of death worldwide, accounting for 7.4 million deaths (around 13% of all deaths) in 2004. Monoclonal/recombinant antibodies, which specifically target clinical biomarkers of disease, have increasingly been applied as powerful tools in cancer imaging and therapy, a fact that is highlighted by some nine FDA-approved monoclonal antibodies (MAbs) or their immunoconjugates (as of December 2008) for use in cancer treatment. In this study, five monoclonal antibodies (MAbs) were generated and characterized against carcinoembryonic antigen (CEA), which is widely used clinically as both a blood and tissue tumor marker of epithelial malignancy. Variable domains (VH and VL) of one the stable MAbs with highest affinity were PCR-amplified and assembled as single-chain antibody fragment (scFv). Following the cloning and expression of scFv antibody fragments in Escherichia coli, the functional binding and specificity of the recombinant antibody were confirmed by ELISA. To develop a direct in vitro detection of CEA-positive cancer cells, scFv DNA was genetically fused to enhanced green fluorescent protein (EGFP) gene and expressed in bacteria. The chimeric fluorescent protein is able to specifically detect CEA-positive cell lines; no cross-reactivity was observed with a negative control cell line. This strategy will likely allow the establishment of a rapid, single-step detection assay of CEA, which is considered to be one of the best predictors of malignancy among all other tumor markers.
    Hybridoma (2005) 06/2011; 30(3):229-38. · 0.33 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Aberrant epidermal growth factor receptor (EGFR) signaling is a common feature of multiple tumor types, including glioblastoma (GBM). As such, EGFR has emerged as an attractive target for antitumor therapy. In the present study, we sought to develop an immunotoxin capable of specifically targeting EGFR-expressing cells and mediating inhibition of cell growth and cell killing. The Luffin P1 (LP1) ribosome inactivating protein was chosen to generate a fusion protein, antiEGFR/LP1, based upon its potent protein synthesis inhibition and small size (5 kDa). LP1 was fused to the C-terminus of an anti-EGFR single-chain antibody (scFv). The recombinant antiEGFR/LP1 protein was expressed in Escherichia coli, and refolded and purified on an immobilized Ni(2+)-affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed that antiEGFR/LP1 was sufficiently expressed. Confocal microscopy and flow cytometry demonstrated that antiEGFR/LP1 bound specifically to EGFR-positive cells (U251), as almost no binding to EGFR-negative (Jurkat cells) was observed under identical time and dosage conditions. Finally, the MTT cell viability assay showed that antiEGFR/LP1 elicited obvious cytotoxicity toward EGFR-positive tumor cells. Collectively, these results suggest that antiEGFR/LP1 is biologically active and specific toward EGFR-positive tumor cells and may represent an effective EGFR-targeted cancer therapy.
    Protein Expression and Purification 02/2012; 83(1):1-7. · 1.43 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In this study, we demonstrate that the biological unnatural amino acid incorporation method can be utilizedin vivo to synthesize an alkyne-terminated telechelic protein. Synthesis of terminally-functionalized polymers such as telechelic polymers is recognized to be important, since they can be employed usefully in many areas of biology and material science, such as drug delivery, colloidal dispersion, surface modification, and formation of polymer network. The introduction of alkyne groups into polymeric material is particularly interesting since the alkyne group can be a linker to combine other materials using click chemistry. To synthesize the telechelic recombinant protein, we attempted to incorporate the L-homopropargylglycine into the recombinant GroES fragment by expressing the recombinant gene encoding Met at the codons for both N- and C-terminals of the protein in the Met auxotrophicE. coli via Hpg supplementation. The Hpg incorporation rate was investigated and the incorporation was confirmed by MALDI-TOF analysis of the telechelic recombinant protein. Keywordsrecombinant protein–telechelic protein–unnatural amino acid–alkynylation
    Macromolecular Research 06/2009; 17(6):424-429. · 1.64 Impact Factor