Botryococcus braunii autolysate for the production of deuterium-labeled recombinant protein

Abstract

Deuterated biomolecules such as proteins, lipids, and DNA are widely used in neutron scattering experiments. This is due to the unique scattering properties of 2H, including a strong positive neutron scattering length while contributing very little background compared to the more abundant 1H isotope. Deuteration is therefore an indispensable component in the study of structure, function, and dynamic behaviour of biomolecules by neutron scattering. In the past we compared multiple microalgae species for their ability to grow under deuterated conditions and in our hands Botryococcus braunii proved the easiest and most resilient to long-term culturing in D2O. In this study we describe how to culture B. braunii cells under deuterated conditions followed by preparation of an aqueous extract. The procedure is based on autolysis where cells are incubated at 50 °C for 24 h and clarified by centrifugation and filtration. The product, deuterated algal autolysate, is then used in minimal media for deuterated recombinant protein production in bacteria. We demonstrate that in-house produced deuterated algal autolysate can fully substitute for glycerol-d8 in minimal media without a reduction in expressed protein yield while obtaining ~98 % deuterium incorporation in the final product, suitable for neutron scattering and other types of experiments.