Expression Characterization And Preparation Of Human Amyloid Precursor Protein (APP) In Escherichia Coli, And The Establishment And Application Of The Cell Models Of Unmodified APP/C99

Alzheimer's disease, commonly known as senile dementia, is the fourth-ranking cause of death in the world. One of the major pathogenesis is believed that amyloid precursor proteins (APP)are abnormally cleaved byγ-secrease. APP is a kind of transmembrane protein throughout human body, but its biological functions have not been clearly identified. There are three isoforms of APP, APP770 (770 amino acids), APP751 (751 amino acids) and APP695 (695 amino acids), and APP695 contributes the most important role in human brain tissue. These APPs can first be cleaved byβ-secretase and remain the C terminal transmembrane protein, C99, which then can be processed toβ-amyloid peptide 42 (Aβ42) with high propensity to aggregate and cytotoxic byγ-secretase in transmembrane region.γ-secretase as a macromolecular complex with multi-subunit internal proteins including Presenilin1(PS1), Anterior pharynx defective (Aph-1), Nicastrin (NCT) and Presenelin enhancer protein 2 (Pen-2), belongs to intramembrane cleaving proteases (I-CLiPs). Among those subunits PS1 is the catalytic component. It is postulated that three phosphorylation sites nearγ-catalytic site may affect the activity ofγ-secretase, but the mechanism and the effect on the production of Aβare still unclear.Both APPs and C99 are integral membrane proteins, yet it has not been reported about the expression of two holo-proteins, APP751 and APP695, and their subcellular location as membrane protein in prokaryotic system. E. coli cell is an appropriate prokaryotic expression host cell for this study to pursue high yield of target protein and, most importantly, to obtain the expression product without post-translational modification. So it is convenient to get abundant unmodified interest proteins (APP770 /APP751/APP695/APP695 MC(from the middle amino acid sequence of APP695 to the C terminal)/C99) from E. coli host cells to analyze the role played by modified or unmodified APP/C99 on the production of Aβin target cells. First, we constructed the expression vectors, APP770/APP751/APP695/APP695MC/C99- pET28a(+), and then determined the optimal expression conditions as follow: at 37℃, induction duration is 4-5 hours, IPTG concentration is 1.0 mM/L, the Glucose concentration is 0.8%(g/v) and the A600=0.6-0.8. After purification by Ni2+ column, about 5 milligrams of purified target protein (APP770/APP751/APP695) can be got from 1 L culture medium. To analyze the subcellular location of these membrane proteins in E. coli, membrane fractions were extracted from the expression bacteria. Treatment of the supernatant of cell lysates with sodium carbonate (pH11.5), membrane-bound protein fractions including APP770/APP751/APP695/APP695MC/C99 can be obtained by ultracentrifugation and were identified by Western blot. It is the first time that the subcellular location of these intramembrane proteins is analyzed, and these results provide convenience for next study.Can the APP/C99-containing membrane fragments from E. coli cells be fused with the cytoplasmic membrane of eukaryotic cells? It is known that fusion of biological membrane happens ubiquitously in the earth, such as endocytosis, exocytosis, transport of vesicle in cells, sperm-ovum interaction, enveloped virus infection of target cells and signal transport of neuron. On the basis of membrane fusion in biological fields, Rice and Harda proved the feasibility of membrane fragments fusing with intact cytoplasmic membrane in 2004. Receptors in the plasma membrane mediate the biological membrane fusion. APP owns the structures of typical membrane receptor. From extracellular side to cytoplasm, APP is composed of N terminal signal peptide, a large extracellular domain (including a cystine rich region, a negative electronics region and N-linked glycosylation sites), a transmembrane domain and a short C terminal region in cytoplasm. It has not been reported whether membranes containning interested intramembrane proteins obtained from prokaryotic cells can be fused with target eukaryotic cell membrane. Membrane-bound APPs (APP770/APP751/APP695/APP695MC/C99) transferred to target cells (PC12/HEK293) could be detected by immunofluorescence and Western blot, and the structure state of membrane fusion could be observed by transmission electron microscope. These results indicated that an unmodified cell models of APP/C99 were established.With the results mentioned above, C99 membrane fragments were fused with the target cells for assessment whether the modification in C99 affected theγ-site proteolysis or the production of Aβand. In PC12 and HEK293 cells, the level of Aβwas all elevated by modified C99 comparing with that by the unmodified C99. This result showed that the modification of C99 could enhance the production of Aβ. So all of the results may provide experimental and theoretical materials for AD treatment.