Isotopic Labeling of Heterologous Proteins in the Yeast Pichia pastoris and Kluyveromyces lactis

Abstract

Several protein expression systems are available for the preparation of stable isotope-labeled recombinant proteins for NMR studies. Yeast expression systems have several advantages over prokaryotic systems, such as the widely used Escherichia coli expression system. Protein expression using the methylotrophic yeast Pichia pastoris is commonly employed for the preparation of isotope-labeled proteins. Recently, the hemiascomycete yeast Kluyveromyces lactis expression system was reported as being useful for preparing proteins for NMR studies. Since each yeast expression system has different features, their applications have increased in number. In this chapter, we describe procedures for the efficient production of uniformly isotope-labeled proteins using the P. pastoris and the K. lactis yeast expression systems.

Copsin, a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis

“…Production of 15 N/ 13 C-labeled Copsin in P. pastoris-The production of isotope-labeled copsin was adapted from a previously published protocol (11). In brief, P. pastoris transformants were inoculated in minimal medium supplemented with 0.5% (w/v) 15 NH 4 Cl (98 atom % 15 N) and 13 C 3 -glycerol (99 atom % 13 C), respectively, and cultivated in shake flasks at 30°C to an A 600 of ϳ35.…”

Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR

“…In the future, this shortcoming may be rectified by using other species of yeast, which can metabolize more complex carbon sources amenable to alternate labeling (Miyazawa-Onami et al, 2013;Nars et al, 2014;Sugiki et al, 2012).…”

“…Possibility of deuteration has been demonstrated as well (Massou et al, 1999;Morgan, Kragt, & Feeney, 2000;Sugiki et al, 2012;Tomida et al, 2003). We further extended these economical shake-flask isotope-labeling protocols (Pickford & O'Leary, 2004;Rodriguez & Krishna, 2001) to eukaryotic membrane proteins and optimized them to produce ssNMR samples giving excellent resolution sufficient for detailed structural characterization (Emami et al, 2013;Fan, Shi, et al, 2011).…”

“…In parallel to that, carbon-and nitrogen-isotope-labeling protocols for soluble proteins for solution NMR are long-established in Pichia (Laroche, Storme, De Meutter, Messens, & Lauwereys, 1994;Pickford & O'Leary, 2004;Sugiki, Ichikawa, Miyazawa-Onami, Shimada, & Takahashi, 2012;Wood & Komives, 1999) and have been optimized to become economical, remaining efficient at the same time (Rodriguez & Krishna, 2001). Possibility of deuteration has been demonstrated as well (Massou et al, 1999;Morgan, Kragt, & Feeney, 2000;Sugiki et al, 2012;Tomida et al, 2003).…”

“…Media Recipes According to the Manual of the Pichia Expression Kit (Invitrogen Life Technologies) and Protocols Used for Solution NMR(Pickford & O'Leary, 2004;Sugiki et al, 2012) Medium CompositionSee text for modifications used for solid-state NMR sample preparation.…”