AN ESCHERICHIA-COLI PLASMID VECTOR SYSTEM FOR PRODUCTION OF STREPTAVIDIN FUSION PROTEINS - EXPRESSION AND BIOSELECTIVE ADSORPTION OF STREPTAVIDIN-BETA-GALACTOSIDASE

Covalently immobilized biotin was used as a biospecific adsorbant to i nvestigate the application of streptavidin as an affinity domain for s imultaneous purification and immobilization of recombinant proteins. A streptavidin-beta-galactosidase fusion protein was constructed and te sted as a model system. The gene for streptavidin from Streptomyces av idinii was modified by polymerase chain reaction to mutate the stop co don and to facilitate cloning into an Escherichia coil expression vect or yielding a versatile plasmid with 37 unique restriction enzyme site s at the 3' end. E. coli beta-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 fusi on protein was expressed. Sulfosuccinimidyl-6-(biotinamido)hexanoate w as covalently immobilized on 3-aminopropyl-controlled-pore glass beads . Exposure of recombinant cell lysates to this support indicated that streptavidin-beta-galactosidase was bioselectively adsorbed. The resul ting biocatalyst contained 300 mg protein per gram of beads and exhibi ted a specific activity of 306 mu mol/min per milligram protein with o -nitrophenyl-beta-D-galactopyranoside as substrate corresponding to ap proximately 50% of that observed for commercially pure E. coli beta-ga lactosidase. (C) 1994 John Wiley & Sons, Inc.