Biodeuteration involves the growth of microorganisms (commonly E. coli) in a heavy water (D2O) culture medium supplemented with either a deuterated or hydrogenated carbon substrate, depending on the level of deuteration required. The biomass is harvested and the deuterated molecule (e.g. protein) is purified and characterised.
The biological deuteration laboratories at the NDF are staffed and equipped to undertake molecular biology, biosynthesis, purification and characterisation of deuterated (2H) and multiple labeled biomolecules (2H, 15N, 13C).
Biodeuteration of molecules is achieved through the use of heavy water (D2O) and/or 2H-labelled carbon sources in the growth medium of the host organism, dependent on the level of deuteration required*. 13C and 15N medium components can be added for multiple labelling of the target biomolecule.
Recombinant expression of proteins is achieved mainly using the microbe Escherichia coli as the host organism, however a yeast host (Pichia sp) can also be employed depending on the target protein properties. Molecular biology expertise is available for insertion of gene sequences into optimal expression vectors for deuteration. The biodeuteration laboratories are also experienced in the production of other biopolymers (e.g. chitosan, polyhydroxyalkanoates, cellulose) in bacteria, yeast and algal systems.
From transformation or seed cultures, small volume flask cultures are grown in shaker incubators and transferred to bioreactors of 1 L to 10 L capacity. The NDF has a bioreactor equipped with photosynthetic light control for algae/photosynthetic microorganism culturing and methanol monitoring and feedback control for yeast growth and protein expression induction.
Purification and characterisation
Labelled biomass is supplied or the target molecules extracted and purified via cell lysis and chromatographic separation or solvent extraction. SDS-PAGE and mass spectrometry analyses confirm the identity of expressed proteins and desired deuteration level of the target protein.
The biological deuteration laboratories have a range of instruments available for purification and characterisation including:
- AKTA chromatography systems (ambient temperature or refrigerated)
- Multi-Angle Light Scattering with dynamic light scattering (Wyatt MALS-QELS)
- Spectrophotometers (including Circular Dichroism)
- Fluorescence plate reader
- Thermal analysis – Differential Scanning Calorimeter (DSC)
- LC-MS/MS (liquid chromatography-tandem mass spectrometer)
- Quartz Crystal Microbalance with Dissipation monitoring (QCM-D)
Examples of labelled biomolecules and collaborative research
Triple-labelled protein (2H/15N/ 13C) and NMR
Investigation of a fungal hydrophobin (EAS∆15) rodlet structure using recombinantly expressed 2H/15N/13C labelled EAS∆15 and solid-state NMR. See example.
Deuterated protein and Neutron Reflectivity
Extracellular matrix proteins such as fibronectin, laminin and collagen can be used to create biomimetic surfaces for cell adhesion and tissue engineering. By immobilising biocompatible ECM proteins on a biomaterial surface the interaction between it and the host cells can be influenced.See example.
Deuterated biopolymer and Infrared Microspectroscopy
Deuteration of bacterial biopolyesters to probe phase separation using the Infrared Microspectroscopy beamline at the Australian Synchrotron. See example.
Dr. Anton Le Brun uses neutron scattering techniques in combination with deuteration to research phenomena that occur at biological membranes.See example.
*NDF Biological Lab Methods for biodeuteration/multiple-labelling of proteins and biodeuteration of biopolymers have recently been published in Methods in Enzymology:
- Duff AP, Wilde KL, Rekas A, Lake V, Holden PJ. Robust high-yield methodologies for 2H and 2H/15N/13C labelling of proteins for structural investigations using neutron scattering and NMR. Methods in Enzymology, 2015, in press. http://dx.doi.org/10.1016/bs.mie.2015.06.014
- Le Brun AP, Clifton LA, Holt SA, Holden PJ, Lakey JH: Deuterium Labeling Strategies for Creating Contrast in Structure–Function Studies of Model Bacterial Outer Membranes Using Neutron Reflectometry. Methods in Enzymology, 2015, in press. http://dx.doi.org/10.1016/bs.mie.2015.05.020
- Russell RA, Garvey CJ, Darwish TA, Foster LJR, Holden PJ. Biopolymer Deuteration for Neutron Scattering and Other Isotope-Sensitive Techniques. Methods in Enzymology, 2015, in press. http://dx.doi.org/10.1016/bs.mie.2015.06.015