An Escherichia coli plasmid vector system for production of streptavidin fusion proteins: Expression and bioselective adsorption of streptavidin-β-galactosidase
✍ Scribed by Marie K. Walsh; Harold E. Swaisgood
- Publisher
- John Wiley and Sons
- Year
- 1994
- Tongue
- English
- Weight
- 625 KB
- Volume
- 44
- Category
- Article
- ISSN
- 0006-3592
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✦ Synopsis
Covalently immobilized biotin was used as a biospecific adsorbant to investigate the application of streptavidin as an affinity domain for simultaneous purification and immobilization of recombinant proteins. A streptavidinp-galactosidase fusion protein was constructed and tested as a model system. The gene for streptavidin from Streptomyces avidinii was modified by polymerase chain reaction to mutate the stop codon and to facilitate cloning into an Escherichia coli expression vector yielding a versatile plasmid with 37 unique restriction enzyme sites at the 3' end. E. coli p-galactosidase was cloned in-frame to the streptavidin gene. Analysis of lysates of induced recombinant E. coli cells by SDS-PAGE and Western blots indicated that the 133.6-kDa fusion protein was expressed. Sulfosuccinimidyl-6-(biotinamido)hex- anoate was covalently immobilized on 3-aminopropylcontrolled-pore glass beads. Exposure of recombinant cell lysates to this support indicated that streptavidin-pgalactosidase was bioselectively adsorbed. The resulting biocatalyst contained 300 mg protein per gram of beads and exhibited a specific activity of 306 Frnol/min per milligram protein with o-nitrophenyl-p-D-galactopyranoside as substrate corresponding to approximately 50% of that observed for commercially pure E. coli p-galactosidase.