title: In vivo uniform (15)N-isotope labelling of plants: using the greenhouse for structural proteomics.
abstract: Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands.
authors.forename: Johannes H
authors.initials: JH
authors.lastname: Ippel
authors.forename: Laurice
authors.initials: L
authors.lastname: Pouvreau
authors.forename: Toos
authors.initials: T
authors.lastname: Kroef
authors.forename: Harry
authors.initials: H
authors.lastname: Gruppen
authors.forename: Geurt
authors.initials: G
authors.lastname: Versteeg
authors.forename: Peter
authors.initials: P
authors.lastname: van den Putten
authors.forename: Paul C
authors.initials: PC
authors.lastname: Struik
authors.forename: Carlo P M
authors.initials: CP
authors.lastname: van Mierlo
mesh_terms: D002247:Carbon Isotopes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes / Q000378:metabolism; D020543:Proteome / Q000737:chemistry* / Q000502:physiology; D040901:Proteomics; D011198:Solanum tuberosum / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D020543:Proteome