title: High-efficient production and biophysical characterisation of nicastrin and its interaction with APPC100.
abstract: Nicastrin, the largest member among the four components of the γ-secretase complex, has been identified to be the substrate recognizer for the proteolytic activity of the complex. Here we report that full-length human nicastrin (hNCT) can be obtained by heterologous expression in E. coli. Milligram quantities of the target protein are purified in a two-step purification protocol using affinity chromatography followed by SEC. The FOS-choline 14 purified tetrameric hNCT exhibits a proper folding with 31% α-helix and 23% β-sheet content. Thermal stability studies reveal stable secondary and tertiary structure of the detergent purified hNCT. A physical interaction between nicastrin and the γ-secretase substrate APPC100 confirmed the functionality of hNCT as a substrate recognizer.
authors.affiliation: Centre for Structural Systems Biology (CSSB), DESY, Hamburg, Germany.|Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Kun
authors.initials: K
authors.lastname: Yu
authors.affiliation: Centre for Structural Systems Biology (CSSB), DESY, Hamburg, Germany.|Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Ge
authors.initials: G
authors.lastname: Yang
authors.affiliation: Centre for Structural Systems Biology (CSSB), DESY, Hamburg, Germany.|Institute of Complex Systems, Structural Biochemistry (ICS-6), Forschungszentrum Jülich, Jülich, Germany.|Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Labahn
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D055592:Biophysical Phenomena; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D016564:Amyloid beta-Protein Precursor; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases