Functional and stable expression of recombinant human FOXP3 in bacterial cells and development of antigen-specific monoclonal antibodies.
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.
Hu
Shuiqing
S
International Joint Cancer Institute, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China.
Dai
Jianxin
J
Wei
Huafeng
H
Fan
Kexing
K
Wang
Huajing
H
Zhang
Dapeng
D
Qian
Weizhu
W
Li
Bohua
B
Wang
Hao
H
Zhu
Tongyu
T
Zhang
Yanyun
Y
Guo
Yajun
Y
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D051858:Forkhead Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D011993:Recombinant Fusion Proteins