Modern Methods for the Expression of Proteins in Isotopically Enriched Form

Abstract

The introduction of stable isotopes into proteins has significantly reduced the time requirements for structure elucidation of biomolecules. Moreover, structural studies of proteins with molecular weights exceeding the 10 kDa limit are usually not possible without uniform isotope labeling because of severe resonance overlap and inefficient coherence transfer along the rather small 3 J 1 H-1 H couplings. Nowadays, efficient expression of recombinant proteins is a prerequisite for many techniques used in structural biology, but the requirement for isotope labeling in particular often precludes NMR (nuclear magnetic resonance) structure determination of proteins isolated from natural sources. Specifically, proteins that have been uniformly labeled with 15 N and/or 13 C are commonly required for NMR spectroscopy, especially for backbone chemical shift assignment procedures, which are greatly facilitated by the use of a series of rather sensitive multi-dimensional triple-resonance NMR experiments (see Chapt. 4) [1], in a process that can also be automated with good success [2]. Moreover, the random replacement of nonexchangeable protons by deuterons reduces 1 H-1 H dipolar interactions and scalar couplings, thereby reducing peak line widths considerably and allowing structure elucidation of proteins exceeding 30 kDa [3,4]. Random fractionally deuterated protein samples also permit the use of longer mixing times in NOESY (nuclear Overhauser effect spectroscopy) experiments, since spin-diffusion pathways are largely eliminated. In addition, transverse-relaxation optimized spectroscopy (TROSY [5], see also Chapt. 10), which has been used for molecules larger than 100 kDa [6], benefits dramatically from deuteration. This stems from the fact that the TROSY component that is narrowed by the DD-CSA compensation is broadened by dipolar interactions with nearby protons.Besides uniform labeling approaches, stable isotopes can also be introduced at specific sites in proteins in order to simplify the assignment process and to isolate spectral information from the region of interest. For example, biosynthetically-directed fractional 13 C labeling offers the possibility of making stereospecific assignments of all isopropyl methyl groups of Val and Leu residues [7]. In another approach often used for solid-state NMR termed residue-specific labeling, isotope labels are introduced at single sites in a protein, as described in another chapter of this volume (Chapt. 11). A related scheme, called amino acid-type labeling, is accomplished by expression in an amino acid-based medium

Preparation of amino‐acid‐type selective isotope labeling of protein expressed in Pichia pastoris

“…Protein labeling plays an important role in structural biology research, and protein structure determination using NMR and X‐ray techniques usually requires labeling of the protein 4. The incorporation level of isotope labeling depends on the expression systems; therefore, the optimization of culture conditions and isotope sources to obtain high levels of isotope incorporation are essential 5–8. For example, there is substantial difference in incorporation levels of isotope labeling previously observed between P. pastoris vs. E. coli .…”

“…Protein labeling is critical for successful structure determination of proteins using NMR spectroscopy and X‐ray crystallography 4. A variety of methods are available to produce isotopically labeled proteins in protein expression systems, including methods for uniform labeling, backbone‐only labeling, methyl‐group labeling, amino‐acid‐type selective (AATS) labeling, segmental labeling, and reverse labeling 5–8…”

Conformational Changes of Troponin C Within the Thin Filaments Detected by Neutron Scattering

“…28,29 Degree of deuteration of the purified dTnC was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy. The relative molecular masses of dTnC and TnC were estimated to be 19,288 and 18,370, respectively.…”

“…Deuterated TnC was obtained from E. coli cells grown in a 2 H 2 O ( 2 H 99.9%, Cambridge Isotope Laboratories, Inc., Andover, MA, USA) medium containing deuterated algal peptone produced from lyophilized cells of the green algae Scenedesmus obliquus grown photoautotrophically in 2 H 2 O. 28,29 Culture was carried out in the presence of ampicillin and chloramphenicol, and expression was induced with IPTG 48 for three hours. The cells were harvested by centrifugation, and after sonication on ice, the cell lysate was removed by centrifugation.…”

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

“…Although knowledge of protein structure is certainly an important factor for the drug discovery process, the predictions at that time regarding the impact of NMR were overly optimistic, primarily due to the fact that the size and solubility of proteins are still the biggest limiting factor for successful NMR analysis. Today, protein structure determination by NMR spectroscopy usually includes isotopic labelling of the protein over-expressed in genetically modified bacteria [3]. Hence, the availability of 13 C, 15 N and 2 H isotopically enriched proteins combined with numerous multidimensional NMR experiments enable sequential resonance assignment and solution state structure determination of proteins or proteinligand complexes up to 82 kDa in size [4].…”