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Baek, Lee, Seo, and Cha: Isolation and Characterization of Human scFv Molecules Specific for Recombinant Human Heat Shock Protein (HSP) 70.1
Original Article

Immune Network 2004; 4(1): 7-15.

Published online: 31 March 2004

DOI: https://doi.org/10.4110/in.2004.4.1.7

Isolation and Characterization of Human scFv Molecules Specific for Recombinant Human Heat Shock Protein (HSP) 70.1

Hyun-jung Baek1, Jae-seon Lee2, Jeong-sun Seo2, Sang-hoon Cha1, 3

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.

Correspondence to: Sang-hoon Cha, Division of Biotechnology, College of Agriculture and Life Science, Kangwon National University, Chunchon 200-701, Korea. (Tel) 82-33-250-6485, (Fax) 82-33-253-6485, chash@kangwon.ac.kr

Copyright © 2004 The Korean Association of Immunologists

(open-access):

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 × 108 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 VH subgroup III. On the other hand, VL of two clones belonged to the kappa light chain subgroup I, but the other utilized Vk 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: Phage display library, scFv, heat shock protein 70, pCANTAB-5E, intrabody

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