Intracellular production of a soluble and functional monomeric streptavidin in Escherichia coli and its application for affinity purification of biotinylated proteins.
Monomeric forms of avidin and streptavidin [(strept)avidin] have many potential applications. However, generation of monomeric (strept)avidin in sufficient quantity is a major limiting factor. We report the successful intracellular production of an improved version of monomeric streptavidin (M4) in a soluble and functional state at a level of approximately 70 mg/L of an Escherichia coli shake flask culture. It could be affinity purified in one step using biotin agarose with 70% recovery. BIAcore biosensor analysis using biotinylated bovine serum albumin confirmed its desirable kinetic properties. Two biotinylated proteins with different degrees of biotinylation (5.5 and 1 biotin per protein) pre-mixed with cellular extracts from Bacillus subtilis were used to examine the use of M4-agarose in affinity purification of protein. Both biotinylated proteins could be purified in high purity with 75-80% recovery. With the mild elution and matrix regeneration conditions, the M4-agarose had been reused four times without any detectable loss of binding capability. The relatively high-level overproduction and easy purification of M4, excellent kinetic properties with biotinylated proteins and mild procedure for protein purification make vital advancements in cost-effective preparation of monomeric streptavidin affinity matrix with desirable properties for purification of biotinylated molecules.
Wu
Sau-Ching
SC
Department of Biological Sciences, University of Calgary, Canada.
Wong
Sui-Lam
SL
D001412:Bacillus subtilis / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019809:Streptavidin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000379:methods
D011994:Recombinant Proteins; D019809:Streptavidin