Université de Neuchâtel

Artificial Metalloenzymes: (Strept)avidin as Host for Enantioselective Hydrogenation by Achiral Biotinylated Rhodium−Diphosphine Complexes

Skander, Myriem ; Humbert, Nicolas ; Collot, Jérôme ; Gradinaru, Julieta ; Klein, Gérard ; Loosli, Andreas ; Sauser, Jérôme ; Zocchi, Andrea ; Gilardoni, François ; Ward, Thomas R.

In: Journal of the American Chemical Society (JACS), 2004, vol. 126, no. 44, p. 14411–14418

We report on the generation of artificial metalloenzymes based on the noncovalent incorporation of biotinylated rhodium−diphosphine complexes in (strept)avidin as host proteins. A chemogenetic optimization procedure allows one to optimize the enantioselectivity for the reduction of acetamidoacrylic acid (up to 96% ee (R) in streptavidin S112G and up to 80% ee (S) in WT avidin)....

Université de Neuchâtel

Artificial Metalloenzymes for Enantioselective Catalysis Based on Biotin−Avidin

Collot, Jérôme ; Gradinaru, Julieta ; Humbert, Nicolas ; Skander, Myriem ; Zocchi, Andrea ; Ward, Thomas R.

In: Journal of the American Chemical Society (JACS), 2003, vol. 125, no. 30, p. 9030-9031

Incorporation of biotinylated racemic three-legged d6-piano stool complexes in streptavidin yields enantioselective transfer hydrogenation artificial metalloenzymes for the reduction of ketones. Having identified the most promising organometallic catalyst precursors in the presence of wild-type streptavidin, fine-tuning of the selectivity is achieved by saturation mutagenesis at...

Université de Neuchâtel

Electrophoretic behavior of streptavidin complexed to a biotinylated probe : A functional screening assay for biotin-binding proteins

Humbert, Nicolas ; Zocchi, Andrea ; Ward, Thomas R.

In: Electrophoresis, 2004, vol. 26, p. 47-52

The biotin-binding protein streptavidin exhibits a high stability against thermal denaturation, especially when complexed to biotin. Herein we show that, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), streptavidin is stabilized at high temperature in the presence of biotinylated fluorescent probes, such as biotin-4-fluorescein, which is incorporated within the binding...

Université de Neuchâtel

Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris

Zocchi, Andrea ; Jobé, Anna Marya ; Neuhaus, Jean-Marc ; Ward, Thomas R.

In: Protein Expression and Purification, 2003, vol. 32, p. 167-174

A recombinant glycosylated avidin (recGAvi) with an acidic isoelectric point was expressed and secreted by the methylotrophic yeast Pichia pastoris. The coding sequence for recGAvi was de novo synthesized based on the codon usage of P. pastoris. RecGAvi is secreted at approximately 330 mg/L of culture supernatant. RecGAvi monomer displays a molecular weight of 16.5 kDa, as assessed...