Immune Netw.
2004 Mar;4(1):7-15. 10.4110/in.2004.4.1.7.
Isolation and Characterization of Human scFv Molecules Specific for Recombinant Human Heat Shock Protein (HSP) 70.1
- Affiliations
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- 1IG Therapy Co., Kangwon National University, Chunchon, South Korea.
- 2ILCHUN Molecular Medicine Institute MRC and Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, South Korea.
- 3Division of Biotechnology, College of Agriculture and Life Science, Kangwon National University, Chunchon, South Korea. chash@kangwon.ac.kr
- KMID: 2054238
- DOI: http://doi.org/10.4110/in.2004.4.1.7
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 X 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.
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