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.
Humbert
Nicolas
N
Institute of Chemistry, University of Neuchatel, Neuchatel, Switzerland.
Schürmann
Peter
P
Zocchi
Andrea
A
Neuhaus
Jean-Marc
JM
Ward
Thomas R
TR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods; D017123:Bacteriophage T7 / Q000378:metabolism*; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification*; D019809:Streptavidin / Q000302:isolation & purification*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019809:Streptavidin; D005947:Glucose