Protocol

Avidin-Biotin Interactions

Volume 418 of the series Methods In Molecular Biology™ pp 101-110

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

  • Nicolas HumbertAffiliated withInstitute of Chemistry, University of Neuchatel
  • , Peter SchürmannAffiliated withInstitute of Chemistry, University of Neuchatel
  • , Andrea ZocchiAffiliated withInstitute of Chemistry, University of Neuchatel
  • , Jean-Marc NeuhausAffiliated withInstitute of Chemistry, University of Neuchatel
  • , Thomas R. WardAffiliated withInstitute of Chemistry, University of Neuchatel

* Final gross prices may vary according to local VAT.

Get Access

summary

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.

Keywords

Streptavidin E. coli affinity purification biotin osmotic stress