Cytoplasmic expression, antibody production, and characterization of the novel zinc finger protein 637.
Zinc finger protein 637 (zfp637), belonging to the Kruppel-like protein family, comprises one atypical C(2)H(2) and six consecutive typical zinc finger motifs. Based on the structural characterization of zfp637 and its location in the cell nucleus, we predict that zfp637 might function as a DNA-binding protein to regulate gene transcription. However, the absence of both a purified zfp637 protein and any commercial antibody for detecting it in cells and tissues has limited functional studies of zfp637 to date. Here, we developed and optimized an expression system by fusing zfp637 with glutathione S-transferase (GST) to achieve a maximal yield of soluble GST-zfp637 fusion protein in Escherichia coli BL21(DE3) cells. The yield was about 10 mg/l of the original bacterial culture. The recombinant GST-zfp637 fusion protein was purified and used for polyclonal antibody production in rabbits. In addition, we developed a method to remove the anti-GST antibody component and obtained a highly purified anti-zfp637 antibody, as demonstrated by an enzyme-linked immunosorbent assay. Western blotting showed that the anti-zfp637 antibody recognized not only the recombinant zfp637 protein but also endogenous zfp637 in several cell lines. The protein was localized mainly in the cell nucleus by immunofluorescence and immunohistochemistry. The expression levels of zfp637 mRNA and protein were significantly increased in NIH3T3 cells treated with 200 μM of H(2)O(2) in a time-dependent manner. The recombinant GST-zfp637 fusion protein and our purified anti-zfp637 antibody will help in elucidating the function of zfp637.
Division of Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, 1# Keyuan 4 Road, Gaopeng Avenue, Hi-tech Development, Chengdu, 610041 Sichuan, People's Republic of China.
Kai
K
Li
Yuyan
Y
Wei
Jie
J
Zhang
Jun
J
Li
Bo
B
Gao
Lugang
L
Huang
Ping
P
Lin
Yuquan
Y
Wei
D000818:Animals; D000917:Antibody Formation / Q000502:physiology*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology*
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C551671:Zfp637 protein, mouse