Nicolas Humbert

High-Yield Production and Purification of Recombinant T7-Tag Mature Streptavidin in Glucose-Stressed E. coli

The overexpression of toxic recombinant proteins is often problematic, leading to either low production levels or inclusion bodies. Streptavidin is no exception and thus the highest production level reported to date for streptavidin is 70 mg/L of functional protein. Herein, we report on the production in Escherichia coli and the purification of a recombinant mature streptavidin bearing a T7-tag. Optimization of critical parameters, including the glucose concentration, the pH and the time of induction as well as the use of BL21(DE3)pLysS cell strain, affords up to 120 mg/L functional streptavidin in soluble form. The yield can be further increased by an osmotic stress during the preculture by adding highly concentrated glucose before the inoculation of the culture medium, thus affording reproducibly 230 mg/L of soluble streptavidin. A single denaturing-renaturing step and affinity chromatography afford highly active tetrameric protein with >3.8/4.0 active sites.

Efficient expression and mutation of avidin and streptavidin as host proteins for enantioselective catalysis

Avidin and structurally related streptavidin are attractive host proteins for the creation of artificial metalloenzymes displaying features reminiscent both of homogeneous catalysts and enzymes. The main advantages are that both proteins have been cloned and expressed in several organisms and possess a deep hydrophobic binding pocket capable of hosting biotinylated catalyst precursors. An optimized artificial avidin gene in Pichia pastoris is expressed. The high level of active protein produced in the extracellular medium is suitable for the performance of high-throughput screening in 96-well plate format. Biologically active recombinant streptavidin is expressed in E. coli. Mutations have been introduced both in avidin and streptavidin genes and both wild type and mutated proteins have been utilized to explore the role of the second coordination sphere in enantioselective catalysis.

Functionality Screen of Streptavidin Mutants by Non-Denaturing SDS–PAGE Using Biotin-4-Fluorescein

Site-directed mutagenesis or directed evolution of proteins often leads to the production of inactive mutants. For streptavidin and related proteins, mutations may lead to the loss of their biotin-binding properties. With high-throughput screening methodologies in mind, it is imperative to detect, prior to the high-density protein production, the bacteria that produce non-functional streptavidin isoforms. Based on the incorporation of biotin-4-fluorescein in streptavidin mutants present in Escherichia coli bacterial extracts, we detail a functional screen that allows the identification of biotin-binding streptavidin variants. Bacteria are cultivated in a small volume, followed by a rapid treatment of the cells; biotin-4-fluorescein is added to the bacterial extract and loaded on an Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) under non-denaturing conditions. Revealing is performed using a UV transilluminator. This screen is thus easy to implement, cheap and requires only readily available equipment.