Protein Expression & Purification

Producing nitrogenase outside its native host requires far more than routine protein expression. These proteins are oxygen-sensitive, metabolically demanding, and often unstable, so sophisticated strategies are essential for obtaining high-quality material in meaningful quantities. By tuning host metabolism, optimizing growth environments, and controlling anaerobic conditions with precision, it is now possible to generate milligram-scale amounts of fully folded, cofactor-containing, and catalytically competent nitrogenase proteins.

This includes reliable expression of NifH, NifDK, NifEN, and NifB, and their accessory factors, as well as the heterologous assembly of the L-, M-, and P-clusters. Tailored workflows, spanning engineered hosts, specialized media, and purification methods that preserve metal integrity, enable these complex proteins to be studied and manipulated outside their native context. Together, these advances provide a robust platform for dissecting nitrogenase biology and exploring new directions in engineering.

Membrane-bound copper transporters are some of the most difficult proteins to express and handle, owing to their strong hydrophobic character, sensitivity to detergents, and dependence on lipid composition. My work focuses on developing strategies to produce and stabilize these transporters in forms suitable for structural and functional studies. This includes CTR-family copper importers from yeast, which require carefully tuned expression hosts and membrane environments, as well as Pco- and Cop-associated outer membrane and periplasmic proteins from E. coli, which participate in copper defense and homeostasis.

By systematically screening detergents, lipids, and buffer systems, and by optimizing expression conditions to support proper folding and metal binding, it is now possible to obtain stable, monodisperse preparations of these transporters for crystallography, biochemical assays, and biophysical characterization. These workflows make it feasible to investigate how copper moves across membranes, how cells defend themselves against copper stress, and how these systems have evolved to balance toxicity with essential biochemical functions.

Notoriously difficult to express, the membrane subunits of respiratory complex I long escaped detailed biochemical study. My work focused on producing these hydrophobic, antiporter-like proteins: NuoL, NuoM, and NuoN, as well as the related MrpA and MrpD components of the Mrp Na⁺/H⁺ antiporter. Success required careful construct design, cytochrome-tag strategies, and expression hosts capable of inserting the proteins correctly into the membrane. By optimizing detergents, lipid additives, and purification conditions, I was able to generate stable, well-behaved preparations suitable for functional assays and structural analysis. These advances enabled direct investigation of how these antiporter-like modules move ions and how their properties link modern complex I to its evolutionary origin in the Mrp antiporter family.