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Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody

Abstract

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, an anti-BAFF single-chain antibody fragment (scFv) was genetically linked to the C terminus of the enhanced green fluorescent protein (EGFP) to generate an EGFP/scFv fusion protein. The EGFP/scFv fusion protein had an apparent molecular weight of 52 kDa and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. After being purified by immobilized metal affinity chromatography, the fusion protein exhibited similar fluorescence spectra with native EGFP. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorting showed the EGFP/scFv could bind to human soluble BAFF and BAFF positive cell lines in vitro. The binding of EGFP/scFv can also be visualized under laser scanning confocal microscopy. Furthermore, the results of the competition assay indicated its antigen binding specificity. Therefore, the fusion protein EGFP/scFv has several characteristics including high sensitivity, stability and convenience for manipulation, and can be a powerful tool for the study of the underlying pathology of BAFF relevant to autoimmune diseases.

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Acknowledgments

This work was supported by the Grants of Nanjing Normal University and Jiangsu Province Graduate Innovation Project (No. 1612005024) and the International Cooperation of Jiangsu Province (No. BZ2007078). We are particularly grateful to Dr. Yuying Feng for the help and technical support in the experiments of fluorescence spectrum, to Dr. ChaoYing Zhang for the help in the experiment of LSCM, and to Dr Ling Wang in Nanjing Medical University for the help and technical support in the experiments of Flow Cytometry Analysis.

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Correspondence to Shuangquan Zhang.

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Cao, M., Cao, P., Yan, H. et al. Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody. Appl Microbiol Biotechnol 79, 423–431 (2008). https://doi.org/10.1007/s00253-008-1447-9

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Keywords

  • Anti-BAFF antibody
  • Enhanced green fluorescent protein (EGFP)
  • Bacterial expression
  • Autoimmune diseases