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Journal of Biochemistry Advance Access originally published online on August 30, 2007
Journal of Biochemistry 2007 142(4):471-480; doi:10.1093/jb/mvm160
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© 2007 The Japanese Biochemical Society.

Functional and Stable Expression of Recombinant Human FOXP3 in Bacterial Cells and Development of Antigen-specific Monoclonal Antibodies

Shuiqing Hu1,3,*, Jianxin Dai1,*, Huafeng Wei1,*, Kexing Fan1, Huajing Wang1, Dapeng Zhang1, Weizhu Qian1,2, Bohua Li1,2, Hao Wang1,2, Tongyu Zhu2, Yanyun Zhang3,{ddagger} and Yajun Guo1,2,3,{dagger}

1International Joint Cancer Institute, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China; 2Shanghai Center for Cell Engineering and Antibody, 399 Libing Road, Shanghai 201203, People's Republic of China; and 3E-Institute of Shanghai Universities Immunology Division, Shanghai Jiao Tong University School of Medicine, 227 South Chongqing Road, Shanghai 200025, People's Republic of China

{dagger}To whom correspondence should be addressed: Tel/Fax: +86-21-25070241, E-mail: gyj{at}zjbio.org

{ddagger}Correspondence may also addressed: Tel: +86-21-63852705, Fax: +86-21-63852705, E-mail: yyzhang{at}sibs.ac.cn

Received June 20, 2007; Accepted July 13, 2007


  Abstract

FOXP3 is a member of the forkhead/winged-helix family of transcriptional regulators which plays a key role in CD4+CD25+ regulatory T-cell (Tregs) function and represents a specific marker for these cells. In order to understand the functional role of FOXP3 and identify Tregs both in normal development and relevant diseases, protein–DNA and protein–protein interaction studies involving this factor are essentially required. Such investigations would be facilitated by the availability of significant amounts of purified FOXP3 protein and specific McAb against FOXP3. Here, we report the purification of human FOXP3 (HFOXP3) expressed in Escherichia coli as a soluble and functional glutathione-S-transferase (GST) fusion protein. Through a single purification procedure, 4 mg of GST-HFOXP3 recombinant protein was obtained per litre of bacterial culture. The biological activity of the recombinant protein was verified by EMSA assay. The yield of folded FOXP3 in the purified GST-FOXP3 was determined by reverse phase HPLC. Besides, we generated and obtained two specific monoclonal antibodies by immunizing BALB/c mouse with the purified GST-HFOXP3 protein. The resulting HFOXP3 protein and anti-HFOXP3 McAbs might provide a useful tool in studying Tregs development and immune regulations.

Key Words: FOXP3, monoclonal antibody, protein expression, purification, Tregs

Abbreviations: EMSA, electrophoretic mobility-shift assay; FOXP3, forkhead box P3; human FOXP3, HFOXP3; McAb, monoclonal antibody; Tregs, regulatory T cells

*These authors contributed equally to this work.


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