[A new method for screening binding proteins that interact with the promoter of lipopolysaccharide inducible genes and its application].
OBJECTIVE
To establish a new method for the study of DNA-protein interaction.


METHODS
Six SPF BALB/c mice were divided into two groups, the lipopolysaccharide (LPS)-treated group (n=3) and the normal control group (n =3). The LPS-treated mice were subjected to tail vein-injection of LPS (25 mg/kg) to reproduce an endotoxic shock model. The biotin-labeled DNA probe for the LPS inducible gene (LIG) promoter was amplified by polymerase chain reaction (PCR). The cell nuclear extracts from liver of mice were prepared and mixed together. DNA pull-down assays were performed by using magnetic beads conjugated with streptavidin. Proteins binding on the beads were eluted by salts with different concentrations. The samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and displayed by silver-stain to compare differential bands between two groups.


RESULTS
Fifteen proteins in the nuclear extracts were found to be different between the mice of two groups. Analysis of the differential bands of SDS-PAGE displayed that, compared with normal control group, there were four proteins increased and one protein declined in the mice treated with LPS when eluted with low salt. While under the condition of high affinity elution, there were nine proteins increased and one disappeared after LPS treatment.


CONCLUSIONS
In this study, a novel method was set up by combining the biotin-streptavidin system and magnetic beads isolation technique, providing a new way to study DNA-protein interaction. This strategy is helpful for further understanding the regulation of gene expression with a view of "omics".
Wang
Juan
J
Department of Pathophysiology and Key Laboratory of Functional Proteomics of Guangdong, Southern Medical University, Guangzhou 510515, Guangdong, China.
Liu
Zhi-feng
ZF
Xu
Jia
J
Yang
Li
L
Li
Zhi-jie
ZJ
Deng
Peng
P
Jiang
Yong
Y
D000818:Animals; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis*; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008070:Lipopolysaccharides / Q000633:toxicity; D008099:Liver / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D011401:Promoter Regions, Genetic; D012772:Shock, Septic / Q000235:genetics* / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry
D015342:DNA Probes; D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D001710:Biotin; D004247:DNA; D019809:Streptavidin