gst融合蛋白亲和色谱法研究转录因子ap2α与tes蛋白的相互作用-study on the interaction between transcription factor ap2α and tes protein by gst fusion protein affinity chromatography.docxVIP

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gst融合蛋白亲和色谱法研究转录因子ap2α与tes蛋白的相互作用-study on the interaction between transcription factor ap2α and tes protein by gst fusion protein affinity chromatography.docx

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gst融合蛋白亲和色谱法研究转录因子ap2α与tes蛋白的相互作用-study on the interaction between transcription factor ap2α and tes protein by gst fusion protein affinity chromatography

N-terminus and three tandem LIM domains in the C-terminus.To study the function of each LIM domain in TES C-terminus individually and identify the true sites interacted with the AP-2α, in this paper, three gene fragments encoding corresponding LIM domains in TES C-terminus were amplified from pCMV-HA-TES plasmid containing the full length of TES coding sequence by PCR and subcloned into prokaryotic expression vector pQE-N1 for constructing expression plasmids pQE-N1-LIM 1, pQE-N1-LIM 2 and pQE-N1-LIM 3. Then the His-tag fusion proteins were expressed in E.coli BL21 and purified with Ni(II)-based immobilized metal ion affinity chromatography.Recombinant E.coli BL21 containing plasmids which included the fused gene fragments encoding AP-2α, expressed fusion proteins with glutathion-S-transferase(GST) in the same way as mentioned above. From the supernatant of the cell sonicates, Glutathion-Sephrose 4B affinity chromatography was employed to isolate the fusion proteins which could be purified to 90% in a single step.Using self-made antibodies against His-TES, the interactions between three LIM domains in TES C-terminus and AP-2α protein fragments were analyzed by affinity chromatography ‘GST pull-down’ assay. The results indicated that the interaction between TES and AP-2α was due to C-terminus of AP-2α binding with the first LIM domains in TES C-terminus. Furthermore, a possible mechanism of self-interaction in TES protein was discussed based on the fact that AP-2α interacted with TES C-terminus instead of TES full-length protein. The self-interaction between LIM domains in TES C-terminus and PET domain in N-terminus could block the binding site of AP-2α in TES LIM domain 1. In this state, TES protein appears as “close” conformation which prohibits it from interacting with AP-2α.Key words: AP-2α; TES gene; LIM domain; affinity chromatography; interactionIV英文缩写表Affinity Chromatography: 亲和色谱Amp: ampicillin 氨苄青霉素AP-2: Activating protein 2 激活蛋白-2bp: base pair 碱基对cDN