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The Korean Association of Immunobiologists (대한면역학회)
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Isolation and Characterization of Human scFv Molecules Specific for Recombinant Human Heat Shock Protein (HSP) 70.1

  • Baek, Hyun-jung (IG Therapy Co., Kangwon National University) ;
  • Lee, Jae-seon (ILCHUN Molecular Medicine Institute MRC and Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine) ;
  • Seo, Jeong-sun (ILCHUN Molecular Medicine Institute MRC and Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine) ;
  • Cha, Sang-hoon (IG Therapy Co., Kangwon National University, Division of Biotechnology, College of Agriculture and Life Science, Kangwon National University)
  • Published : 2004.03.31
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Abstract

Background: The heat shock proteins (HSPs) play an important role in cellular protection mechanisms against physical or chemical stresses. In this study scFv antibodies specific for human HSP70.1 were isolated from a semi-synthetic human scFv library with the ultimate goal of developing anti-HSP70.1 intracellular antibody (intrabody) that may offer an attractive alternative to gene targeting to study the function of the protein in cells. Methods: A semi-synthetic human scFv display library ($5{\times}10^{8}$ size) was constructed using pCANTAB-5E vector and the selection of the library against bacterially expressed recombinant human HSP70.1 was attempted by panning. Results: Three positive clones specific for recombinant HSP70.1 were identified. All three clones used $V_{H}$ subgroup III. On the other hand, $V_{L}$ of two clones belonged to the kappa light chain subgroup I, but the other utilized $V_{k}$ subgroup IV Interestingly, these scFv molecules specifically reacted to the recombinant HSP70.1, yet failed to recognize native HSP70 induced in U937 human monocytic cells by heat treatment. Conclusion: Our results indicated that affinity selection of an scFv phage display library using recombinant antigens produced in E. coli might not guarantee the isolation of scFv antibody molecules specific for a native form of the antigen. Therefore, the source of target antigens needs to be chosen carefully in order to isolate biofunctional antibody molecules.

Keywords

Acknowledgement

Supported by : Korea Research Foundation

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