[Expression and purification of a recombinant transmembrane domain amyloid precursor protein associated with Alzheimer's disease].
More than half of the mutations of the amyloid precursor protein (APP) discovered in familiar forms of Alzheimer's disease are located in the transmembrane domain. The pathogenic mutations presumably affect the lateral dimerization of the APP transmembrane domain in the membrane and change the dimer conformation and/or stability. Thus, the mutations cause an alternative APP digestion pattern in the membrane and neurotoxic amyloid beta-peptide generation. For the detailed study of the specific protein-protein and protein-lipid interactions of the APP transmembrane domain, an E. coli recombinant expression construct was made. The recombinant protein contains an APP transmembrane domain (APPtm(686-726)) with adjacent extramembrane N and C ends. Here, we report the method of isotope-labeled APPtm expression and purification in quantities necessary for a heteronuclear NMR spectroscopy structure and dynamics study. On the basis of the (1)H-(15)N-HSQC spectra, we developed APPtm(686-726) solubilization conditions in the membrane-emulated milieu detergent micelles and lipid bicelles.
Bocharova
O V
OV
Nadezhdin
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KD
Bocharov
E V
EV
Arsen'ev
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D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004134:Dimyristoylphosphatidylcholine / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008567:Membranes, Artificial; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular; D010713:Phosphatidylcholines; D010767:Phosphorylcholine / Q000031:analogs & derivatives; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D016564:Amyloid beta-Protein Precursor; D008567:Membranes, Artificial; D008823:Micelles; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D010767:Phosphorylcholine; C032166:1,2-hexanoylphosphatidylcholine; C028810:dodecylphosphocholine; C064744:dimyristoylphosphatidylsulfocholine; D004134:Dimyristoylphosphatidylcholine