清道夫受体介导的脂质蓄积的分子机制课件（PPT 42页）.ppt

(6) Construction of expression plasmids (8) DiI- AcLDL uptake assay Acknowledgement Yaoyu Chen Xiaohua Wang Jingjing Ben Hui Bai Leming Fan Yong Ji Xiuying Chen * Di-leucine signal motif in the cytoplasmic domain is required for MSR-A-mediated internalization. * Di-leucine signal motif-related endocytosis of Ac-LDL may depend on the clathrin-coat pit. * There might be multiple signal motives in cytoplasmic domain of MSR, which adapts the multiple functions of MSR. Di-Leucine motif may be an distinct motif from the VXFD in SR-A mediated internalization and adhesion. 2. Identification of peptide ligands for the cytoplasmic domain of SRA (1) GST -cSRA fusion protein expression and purification: GST fusion protein were separated on a 12% SDS. 1, purified GST fusion protein; 2, E. coli supernatant containing GST fusion protein; 3 and 6, E. coli supernatant; 4, molecular weight standards; 5, E. coli supernatant containing GST. 1 2 3 4 5 6 Western-blot of the fusion protein with anti-GST antibody. 1, GST-cytoplasmic domain of SRA fusion protein;2, GST; 3, E. coli supernatant; 4, molecular weight standards. 1 2 3 4 (2) Screening the phage peptide library with the fusion protein : 1.3×10-5 3.25×106 2.5×1011 4 2.33×10-6 6.4×105 2.75×1011 3 8.11×10-6 3.45×104 4.25×1011 2 1.34×10-8 5.9×103 4.4×1011 1 Relative output ratio** Output phages（pfu） Input phages（pfu*） Bio panning rounds The pool of phage becomes enriched in favour of sequences that bind to the target after four rounds of bio-panning * phage form unit ** output phages/input phages Binding specificity of the positive phage clones analysed by ELISA using anti-M13 antibody. The bl