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One kind of photosensitizer binding protein (PBP) screening , prokaryotic expression and purification of affinity with the photosensitizer and functional studies

Author: GuoYanRong
Tutor: WeiXunBin；ZhuNaiShuo
School: Fudan University
Course: Microbiology
Keywords: Photodynamic therapy Photosensitizer Photosensitizer binding protein Eukaryotic translation elongation factor 1 alpha 1(eEF1A1)
CLC: Q789
Type: Master's thesis
Year: 2011
Downloads: 73
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Abstract

ALA-PDT has been used primarily in the treatment of non-melanoma skin cancers or those accessible organs, such as the bladder, oral cavity, lungs, upper and lower gastrointestinal tract, and cervix with optical devices. And PpⅨfluorescence has been extensively used in the detection of early malignancies, carcinomas in situ, and cancer precursor lesions and clinical PDT treatment in tumor lesions. However, the exact mechanism of PpIX selective accumulation in tumor cell is not clear. In our previous study, we research the mechanism of PpIX selective accumulation in tumor cell by screening photosensitizer binding protein to find the potential PDT target.We successfully screened a cDNA clone coding a protein binding to PpIX, one of the most widely used compounds in PDT, from phage displayed cDNA library, which is widely used for ligand screening. DNA sequencing result showed that along with phage DNA, this cDNA had open reading frame encoding 25 amino acids, which is eukaryotic translation elongation factor 1 alphal(eEF1A1) C terminal lysine motif (LysM). Specifically interacted with photosensitizer, the eEFlA1 C terminal LysM was called photosensitizer binding peptide (PBPe) and those which included more than 70 percent of PBPe sequence such as eEF1A1 were called photosensitizer binding protein (PBPr).In the present study, PBPr gene was cloned into prokaryotic expression vector pet22b(+) and transformed into E. coli strain BL21 (DE3). The protein gene was placed downstream of N-terminal pelB signal sequence for potential periplasmic localization. After inducted expression by IPTG, the protein was extracted and purified by His-Trap Crude column chromatography. Subsequently eEFlAl was verified by Western blot and MS. Meanwhile, we artificially synthesized eEF1A1 C terminal LysM and verified by HPLC and MS. We demonstrated that both PBPr and PBPe have the ability to bind specifically with PpIX with ELISA and calculated their affinity constants respectively with PpIX using Biacore biomolecule interaction assay. The result above showed that eEF1A1 protein and its C terminal LysM specifically bind to the photosensitizer PpIX directly in vitro.To study intracellular roles of PBP, eEF1A1 full-length, its sub-unit which deleted its C terminal LysM and eEFlAl C terminal LysM, which were successively called A, B and C for short, were cloned respectively to eukaryotic expression vector pEGFP-C 1 to form GFP-fusion protein expression plasmid. After transient transfected with the plasmid mentioned above, the PBP high expression tumor cells by adding 5-ALA, were observed more PpIX accumulation under confocal microscope. In addition, the GFP positive cells were selected from transient transfected tumor cells by Flow cytometry using GFP fluorescent probes and cultivated for a while. The PBP high expression tumor cells by adding 5-ALA, were measured by M5. The result showed that PBP can enhance PpⅨselectively accumulation in tumor cell in vivo.Expect for its central role in protein biosynthesis, eEF1A1 had been reported for more possible roles in apoptosis and protein degradation. The eEF1A1 and eEF1A1 C terminal LysM as 5-ALA & PpⅨPDT targets may reveal a novel mechanism of PDT.

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