title: Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems.
abstract: Extracellular lipase Lip2 from Yarrowia lipolytica is a promising biocatalyst with unusual structural features, as indicated by X-ray crystallography. These features comprise a mobile domain called the lid that controls access to the catalytic site. Conformational rearrangements of the lid have been suggested to regulate lipase enzymatic activities. We used nuclear magnetic resonance to investigate the dynamics of Lip2 by exploring four expression systems, Escherichia coli, cell-free, Pichia pastoris and Y. lipolytica to produce uniformly labelled enzyme. The expression of Lip2 was assessed by determining its specific activity and measuring (15)N-(1)H HSQC spectra. Y. lipolytica turned out to be the most efficient expression system. Here, we report the first use of Y. lipolytica as an expression host for the production of uniform stable isotopic labelled protein for further structural and dynamics studies using NMR.
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France; CNRS, IPBS UMR 5089, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, BP 64182, F-31077 Toulouse Cedex 04, France.
authors.forename: G
authors.initials: G
authors.lastname: Nars
authors.affiliation: CNRS, IPBS UMR 5089, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, BP 64182, F-31077 Toulouse Cedex 04, France.
authors.forename: O
authors.initials: O
authors.lastname: Saurel
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: F
authors.initials: F
authors.lastname: Bordes
authors.affiliation: CNRS, IPBS UMR 5089, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, BP 64182, F-31077 Toulouse Cedex 04, France.
authors.forename: I
authors.initials: I
authors.lastname: Saves
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: M
authors.initials: M
authors.lastname: Remaud-Siméon
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: I
authors.initials: I
authors.lastname: André
authors.affiliation: CNRS, IPBS UMR 5089, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, BP 64182, F-31077 Toulouse Cedex 04, France.
authors.forename: A
authors.initials: A
authors.lastname: Milon
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France. Electronic address: alain.marty@insa-toulouse.fr.
authors.forename: A
authors.initials: A
authors.lastname: Marty
mesh_terms: D020134:Catalytic Domain; D002474:Cell-Free System / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D007553:Isotope Labeling / Q000379:methods*; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase
keywords: Cell-free; Isotope labelling; Pichia pastoris; Yarrowia lipolytica; Yarrowia lipolytica lipase lip2; Yeast