title: Bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human APP binding protein LR11/SorLA for NMR studies.
abstract: LR11 (SorLA) is a recently identified neuronal protein that interacts with amyloid precursor protein (APP), a central player in the pathology of the Alzheimer's disease (AD). AD is a neurodegenerative disease and the most common cause of dementia in the elderly. Current estimates suggest that as many as 5.3 million Americans are living with AD. Recent investigations have uncovered the pathophysiological relevance of APP intracellular trafficking in AD. LR11 is of particular importance due to its role in regulating APP transport and processing. LR11 is a type I transmembrane protein and belongs to a novel family of Vps10p receptors. Using a new expression vector, pMTTH (MBP-MCS1 (multiple cloning site)-Thrombin protease cleavage site-MCS2-TEV protease cleavage site-MCS3-His(6)), we successfully expressed, purified and reconstituted the LR11 transmembrane (TM) and cytoplasmic (CT) domains into bicelles and detergent micelles for NMR structural studies. This new construct allowed us to overcome several obstacles during sample preparation. MBP fused LR11TM and LR11TMCT proteins are preferably expressed at high levels in Escherichia coli membrane, making a refolding of the protein unnecessary. The C-terminal His-tag allows for easy separation of the target protein from the truncated products from the C-terminus, and provides a convenient route for screening detergents to produce high quality 2D (1)H-(15)N TROSY spectra. Thrombin protease cleavage is compatible with most of the commonly used detergents, including a direct cleavage at the E. coli membrane surface. This new MBP construct may provide an effective route for the preparation of small proteins with TM domains.
authors.affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University, Hershey, PA 17033, USA.
authors.forename: Xingsheng
authors.initials: X
authors.lastname: Wang
authors.forename: Richard L
authors.initials: RL
authors.lastname: Gill
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhu
authors.forename: Fang
authors.initials: F
authors.lastname: Tian
mesh_terms: D000368:Aged; D000544:Alzheimer Disease / Q000378:metabolism; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D005822:Genetic Vectors; D006639:Histidine / Q000378:metabolism; D006801:Humans; D026502:LDL-Receptor Related Proteins / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008823:Micelles; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
chemical_list: D016564:Amyloid beta-Protein Precursor; D003902:Detergents; C471213:His-His-His-His-His-His; D026502:LDL-Receptor Related Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008823:Micelles; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C102582:SORL1 protein, human; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease; D013917:Thrombin