Development and application of an economic approach to isotope labeling in higher eukaryotes for NMR studies

Opitz, Christian. Development and application of an economic approach to isotope labeling in higher eukaryotes for NMR studies. 2016, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12646

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Cutting-edge structural studies by crystallography are presently aimed at very complex targets including membrane, multi-domain and secreted proteins of human origin. Such proteins can often only be expressed in higher eukaryotic systems, such as insect or mammalian cell lines due to the requirement of the complex eukaryotic protein expression and post-translational modification machinery. For NMR studies, the need of isotope labeling poses extreme challenges in such eukaryotic hosts, since the growth media need to contain isotope-labeled amino acids. The costs of commercially available isotope-labeled amino acids are very high and so far even prohibitive in the case of deuteration.
This thesis describes the development of an economic approach to isotope labeling in higher eukaryotes, where the necessary labeled amino acids are provided to the growth media from autolyzed yeast grown on inexpensive 13C glucose, 15N ammonium salt and 2H2O. The method was used in the baculovirus-infected insect cell system to produce isotope-labeled proteins relevant to human health and their study by NMR.
Chapter 1 provides an introduction to recombinant expression in insect cell lines and the use of the baculovirus expression vector system. Furthermore, the amino acid metabolism in insect cells and its relevance for isotope labeling is discussed. Frequently used approaches to isotope labeling and their applications are outlined.
Chapter 2 describes in detail the production of isotope-labeled yeast extracts and their supplementation to insect cell growth media for labeled protein expression. Parts of this chapter have been published (Opitz et al., An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts. J. Biomol. NMR, 2015). The method achieves uniform protein 15N and 13C labeling of up to 90 % and deuteration levels of >60 %, which are the highest reported incorporation levels based on cellular extracts so far.
Chapter 3 describes the design of a thermostabilized mutant of the turkey β1-adrenergic receptor (TS-β1AR) for NMR solution studies. This mutant was expressed with selective 15N-valine labeling in insect cells and used for an extensive NMR characterization of ligand interactions. It was possible to follow ligand-induced backbone motions at many key residues in the receptor. The latter results have been published (Isogai, Deupi, Opitz et al., Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor. Nature, 2016). The final section of the chapter shows the expression of uniformly 2H-15N labeled TS-β1AR, which has been used to record high-quality 15N-edited NOESY spectra and obtain a number of further backbone resonance assignments.
Chapter 4 describes the expression of the human lipopolysaccharide-binding protein (hLBP) for NMR studies in baculovirus-infected insect cells by secretion of the protein to the culture medium. A labeling scheme was explored based on the combination of isotope-labeled autolyzed yeast and commercial extracts of algal cells. The combined approach made it possible to produce milligram quantities of uniformly 15N-labeled hLBP and to acquire two-dimensional 1H-15N-TROSY spectra.
Chapter 5 describes the enzymatic synthesis of 15N2-L-glutamine from inexpensive 2-oxoglutaric acid and 15N ammonium salt. Since supplementation of L-glutamine is required for efficient expression, the method further reduces the costs of the labeling approach based on autolyzed yeast and enables economic production of selectively deuterated L-glutamine. For additional cost reduction, also the recombinant expression of glutamate-ammonia ligase was established.
Chapter 6 describes the use of autolyzed yeast as an economical source of labeled amino acids in the cell-free expression system. An initial protocol for high-yield expression was established and its optimization is discussed. Isotope labeling by 15N and 2H is demonstrated for the green fluorescent protein GFP produced in the cell-free expression system based on isotope-labeled yeast.
Chapter 7 describes a proteomics approach to study short-term adaptation of E. coli to growth on 2H7-glucose and 2H2O. Characterization of the cellular adaptation process has implications for the production of deuterated proteins. The results show that deuterated media induce differential expression of proteins involved in the response to osmotic and temperature-induced stress. The data further indicate downregulation of proteins involved in ribosome biogenesis and differential activation of chaperones targeting nucleic acids as well as proteins. Simultaneously, an alteration of the energy metabolism was observed in the perdeuterated environment, in particular upregulation of anaplerotic reactions in the TCA cycle.
Chapter 8 explores the use of isotope-labeled yeast as an economic source of amino acids for labeling in mammalian cells. The initial results indicate that, similar to the protocol applied in insect cells, inexpensive dropout media supplemented by labeled yeast extracts enable good expression of the uniformly labeled angiotensin II receptor type 1a for NMR studies.
Advisors:Grzesiek, Stephan and Maier, Timm
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Grzesiek)
UniBasel Contributors:Opitz, Christian and Grzesiek, Stephan and Maier, Timm
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12646
Thesis status:Complete
Number of Pages:1 Online-Ressource (xvi, 181 Seiten)
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Last Modified:27 Jun 2018 04:30
Deposited On:26 Jun 2018 14:17

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