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An Escherichia coli plasmid vector system for production of streptavidin fusion proteins: Expression and bioselective adsorption of streptavidin‐β‐galactosidase
Author(s) -
Walsh Marie K.,
Swaisgood Harold E.
Publication year - 1994
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260441111
Subject(s) - streptavidin , fusion protein , escherichia coli , recombinant dna , biotinylation , expression vector , chemistry , microbiology and biotechnology , biochemistry , lac operon , plasmid , biotin , biology , gene
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 streptavidin‐β‐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 β‐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) hexanoate was covalently immobilized on 3‐aminopropyl‐controled‐pore glass beads. Exposure of recombinant cell lysates to this support indicated that streptavidin‐β‐galactosidase was bioselectively adsorbed. The resulting biocatalyst contained 300 mg protein per gram of beads and exhibited a specific activity of 306 βmol/min per milligram protein with o ‐nitrophenyl‐β‐ D ‐galactopyranoside as substrate corresponding to approximately 50% of that observed for commercially pure E. coli β‐galactosidase. © 1994 John Wiley & Sons, Inc.