title: Cell-free expression of the APP transmembrane fragments with Alzheimer's disease mutations using algal amino acid mixture for structural NMR studies.
abstract: Structural investigations need ready supply of the isotope labeled proteins with inserted mutations n the quantities sufficient for the heteronuclear NMR. Though cell-free expression system has been widely used in the past years, high startup cost and complex compound composition prevent many researches from the developing this technique, especially for membrane protein production. Here we demonstrate the utility of a robust, cost-optimized cell-free expression technique for production of the physiologically important transmembrane fragment of amyloid precursor protein, APP686-726, containing Alzheimer's disease mutations in the juxtamembrane (E693G, Arctic form) and the transmembrane parts (V717G, London form, or L723P, Australian form). The protein cost was optimized by varying the FM/RM ratio as well as the amino acid concentration. We obtained the wild-type and mutant transmembrane fragments in the pellet mode of continuous exchange cell-free system consuming only commercial algal mixture of the (13)C,(15)N-labeled amino acids. Scaling up analytical tests, we achieved milligram quantity yields of isotope labeled wild-type and mutant APP686-726 for structural studies by high resolution NMR spectroscopy in membrane mimicking environment. The described approach has from 5 to 23-fold cost advantage over the bacterial expression methods described earlier and 1.5 times exceeds our previous result obtained with the longer APP671-726WT fragment.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia. Electronic address: o.bocharova@gmail.com.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Bocharova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia; Moscow Institute of Physics and Technology (State University), 9 Institutskiy per., 141700, Dolgoprudny, Russia.
authors.forename: Anatoly S
authors.initials: AS
authors.lastname: Urban
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia; Moscow Institute of Physics and Technology (State University), 9 Institutskiy per., 141700, Dolgoprudny, Russia.
authors.forename: Kirill D
authors.initials: KD
authors.lastname: Nadezhdin
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia; National Research Centre "Kurchatov Institute", Dept. Complex NBIC-technologies, Akad. Kurchatova pl. 1, 123182, Moscow, Russia.
authors.forename: Eduard V
authors.initials: EV
authors.lastname: Bocharov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia; Moscow Institute of Physics and Technology (State University), 9 Institutskiy per., 141700, Dolgoprudny, Russia.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Arseniev
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000235:genetics*; D002474:Cell-Free System / Q000378:metabolism*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: C545487:APP protein, human; D000596:Amino Acids; D016564:Amyloid beta-Protein Precursor; D011994:Recombinant Proteins
keywords: Algal mixture; Alzheimer's disease mutations; Amyloid precursor protein; Cell-free expression system; NMR spectroscopy; Transmembrane domain