title: Design and characterization of a modular membrane protein anchor to functionalize the moss Physcomitrella patens with extracellular catalytic and/or binding activities.
abstract: Heterologous enzymes and binding proteins were secreted by the moss Physcomitrella patens or anchored extracellularly on its cell membrane in order to functionalize the apoplast as a biochemical reaction compartment. This modular membrane anchoring system utilizes the signal peptide and the transmembrane segment of the somatic embryogenesis receptor-like kinase (SERK), which were identified in a comprehensive bioinformatic analysis of the P. patens genome. By fusing the soluble enzyme NanoLuc luciferase to the signal peptide, its secretion capability was confirmed in vivo. The membrane localization of hybrid proteins comprising the SERK signal peptide, NanoLuc or other functional modules, the SERK transmembrane anchor, and a C-terminal GFP reporter was demonstrated using fluorescence microscopy as well as site-specific proteolytic release of the extracellular enzyme domain. Our membrane anchoring system enables the expression of various functional proteins in the apoplast of P. patens, empowering this photoautotrophic organism for biotechnological applications.
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München , 85350 Freising-Weihenstephan, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Morath
authors.forename: Dong-Jiunn Jeffery
authors.initials: DJ
authors.lastname: Truong
authors.forename: Florian
authors.initials: F
authors.lastname: Albrecht
authors.forename: Ingmar
authors.initials: I
authors.lastname: Polte
authors.forename: Rosario Adriano
authors.initials: RA
authors.lastname: Ciccone
authors.forename: Louise Friederike
authors.initials: LF
authors.lastname: Funke
authors.forename: Leonie
authors.initials: L
authors.lastname: Reichart
authors.forename: Christopher Guy
authors.initials: CG
authors.lastname: Wolf
authors.forename: Andreas-David
authors.initials: AD
authors.lastname: Brunner
authors.forename: Katrin
authors.initials: K
authors.lastname: Fischer
authors.forename: Philipp Constantin
authors.initials: PC
authors.lastname: Schneider
authors.forename: Johanna Barbara
authors.initials: JB
authors.lastname: Brüggenthies
authors.forename: Fabian
authors.initials: F
authors.lastname: Fröhlich
authors.forename: Gertrud
authors.initials: G
authors.lastname: Wiedemann
authors.forename: Ralf
authors.initials: R
authors.lastname: Reski
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019068:Bryopsida / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases