[Prokaryotic expression, purification, and polyclonal antibody generation of beta subunit of human F1F0-ATP synthase].
OBJECTIVE
To express and purify the beta subunit of human F1F0-ATP synthase (hATP5B) in prokaryotic system, and generate its polyclonal antibody in rabbits.


METHODS
The coding sequence of hATP5B mature peptide was amplified by RT-PCR from human umbilical vein endothelial cells (HUVEC) and then cloned into prokaryotic expression vector pET28a(+). The plasmid was transformed into E.coli BL21(DE3) to express hATP5B in reponse to IPTG induction. Expressed protein was purified by Ni(2+) metal-chelating chromatograph, and refolded by dialysis. The products were analyzed by SDS-PAGE and Western blot. Refolded protein was injected into rabbits to generate polyclonal antibody. The titer of the polyclonal antibody was determined by indirect ELISA. The specificity and the binding ability were detected by Western blot and cell immunofluorescence analysis.


RESULTS
DNA sequencing confirmed that the coding sequence of hATP5B mature peptide was completely concordant with the original sequence (NM_001686, hATP5B's GenBank accession number). SDS-PAGE showed that the relative molecular masses (M(r)) of the expressed, purified, and refolded products were about relative molecular masses M(r) 55,000, which was in accordance with the predicted. Grayscale scanning showed that the expressed recombinant hATP5B (rhATP5B) accounted for 36.8% of the total bacteria protein, and the purity of purified product was 98.3%, of refolded 99.1%. The result of Western blot is positive. The titer of the polyclonal antibody was 1:640,000, and it specifically recognized the native antigen in HUVEC.


CONCLUSIONS
hATP5B expressed in prokaryotic system has strong immunogenicity, and the polyclonal antibody with high titer and specificity was obtained from immunization of rabbits. The high level prokaryotic expression of hATP5B and the preparation of its antibody lay the foundation for further function research of hATP5B.
Yu
Li-li
LL
Department of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China. nanshi_yulili@sina.com
Zhang
Yuan-jun
YJ
Zhang
Xia
X
Wang
Yi-bo
YB
Lin
Jian-bo
JB
Ni
Jian
J
Peng
Yan
Y
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000906:Antibodies; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases