1. Design and characterization of a modular membrane protein anchor to functionalize the moss Physcomitrella patens with extracellular catalytic and/or binding activities.
- Author
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Morath V, Truong DJ, Albrecht F, Polte I, Ciccone RA, Funke LF, Reichart L, Wolf CG, Brunner AD, Fischer K, Schneider PC, Brüggenthies JB, Fröhlich F, Wiedemann G, Reski R, and Skerra A
- Subjects
- Amino Acid Sequence, Binding Sites, Bryopsida genetics, Bryopsida metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Molecular Sequence Data, Plant Proteins genetics, Plant Proteins metabolism, Protein Kinases chemistry, Protein Kinases genetics, Protein Kinases metabolism, Protein Sorting Signals, Protein Structure, Tertiary, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Bryopsida chemistry, Membrane Proteins chemistry, Plant Proteins chemistry, Protein Engineering methods, Recombinant Fusion Proteins chemistry
- 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.
- Published
- 2014
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