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.
Humbert
Nicolas
N
Institute of Chemistry, University of Neuchatel, Neuchatel, Switzerland.
Ward
Thomas R
TR
D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005452:Fluoresceins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D019809:Streptavidin / Q000235:genetics*
D005452:Fluoresceins; C118194:biotin-4-fluorescein; D001710:Biotin; D019809:Streptavidin