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Identification of the Interacting Protein of HSF4b Through GST Pull-down Assay

Author: GanGuanQi
Tutor: LiuMuGen
School: Huazhong University of Science and Technology
Course: Genetics
Keywords: Cataract HSF4b protein-protein interaction ZNF143 GST pull-down
CLC: R776.1
Type: Master's thesis
Year: 2011
Downloads: 241
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Abstract

Cataract is one of the most serious eye disease and is the most common reason of human blindness. Heat shock transcription factor 4 belong to HSFs family, the family members activate heat-shock response genes under conditions of heat or other stresses to regulate normal cell growth and development process. HSF4 is associated with cataract, its mutations can cause congenital cataracts and age-related cataract. Two alternatively spliced transcripts encoding distinct isoforms (HSF4a and HSF4b) and possessing different transcriptional activity have been described. HSF4b highly expressed in the lens and is considered highly correlated with cataract. Although several different research groups prepared knock-out mice, we do not know clearly the molecular mechanisms by which HSF4 cause cataract.Detecting HSF4 interacting protein is important to reveal the molecular mechanism by which its mutations cause cataract. Our lab got whole length cDNA of HSF4b from human lens epithelial cell RNA by RT-PCR. Our laboratory cloned the whole length cDNA sequence of HSF4b and lens cell DNA library have been constructed. We detected some proteins that HSF4b may interacts with from a yeast two-hybrid, including the zinc finger transcription factor 143 (ZNF143), white-blue plaque selection have conformed the interaction of these two genes.Here we first conformed whether ZNF143 has the interaction with HSF4b. We constructed a prokaryotic expression plasmid of GST-HSF4b fusion protein. Then we transformed and expressed the GST-HSF4b fusion protein in Escharichia coli strain BL21, purified the fusion protein through glutathione-sepharose immunity affinity. After that, we identified the protein-protein interaction between ZNF143 and HSF4b by GST pull-down assay.At last, we also detected the unkown interacting proteins of HSF4b by GST pull-down assay combined with SDS-PAGE sliver staining analysis. After GST pull-down, we got 2 candidate protein bands on the silver staining SDS-PAGE gels.In conclusion, we used GST pull-down assay here to identify the interacting proteins of HSF4b revealed in the yeast two-hybrid systerm. Then wo derectly detect the interacting proteins of HSF4b throuth GST pull-down assay. Our studies provide new clues for HSF4b new function research, and the further study may provide the basis for revealing the HSF4b function and clarifying the pathogenesis of congenital cataracts.

Identification of the Interacting Protein of HSF4b Through GST Pull-down Assay

Abstract

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