Expression System for Enhanced Green Fluorescence Protein Conjugated Recombinant Antibody Fragment

ArticleinHybridoma and Hybridomics 23(5):279-86 · November 2004with18 Reads
DOI: 10.1089/hyb.2004.23.279 · Source: PubMed

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.

    • "To quantify the binding activity of scFv against c-Met, ELISA was performed as described previously (Yi et al., 2004). Briefly, 10 mg of soluble fraction was added to the plate coated with c-Met and incubated at room temperature for 1 h. "
    [Show abstract] [Hide abstract] ABSTRACT: Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti-c-Met scFv, which was a cytoplasmic non-foldable protein, was redesigned as the model system. This study confirmed that the engineered anti-c-Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c-Met antigen. The structures of the wild-type anti-c-Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition.
    Full-text · Article · Jun 2010 · Biotechnology and Bioengineering
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  • [Show abstract] [Hide abstract] ABSTRACT: c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction between Ni2+ and histidine.
    No preview · Article · Jun 2006 · Protein Expression and Purification
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  • [Show abstract] [Hide abstract] ABSTRACT: Objective: To express the fusion protein of enhanced green fluorescent protein (EGFP) with "ZZ" peptide of staphylococcal protein A (SPA) in E. coli and to test its bioactivity. Methods: The fragment of EGFP gene was cloned into pEZZ 18 vector containing ZZ peptide gene. The recombinant plasmid was transferred into E. coli HB101. The fusion protein was expressed in E. coli and its bioactivity was examined by competitive ELISA and fluorescence properties. Results: The plasmid pEZZ-EGFP correctly expressed in E. coli. The fusion protein retains the bifunctional effects of "ZZ" peptide and EGFP. Conclusion: The fusion protein of ZZ peptide wiui EGFP was expressed as a signal protein. ZZ peptide selectively binds to immunoglobulins G (IgG) of many mammalian, via Fc region of IgG. So the fusion protein could be applied to immunoassay, as a new alternative universal reagent.
    No preview · Article · Jun 2007
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