Protein engineering of streptavidin for in vivo assembly of streptavidin beads.
Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.
Peters
Verena
V
Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Rehm
Bernd H A
BH
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000906:Antibodies / Q000032:analysis; D001665:Binding Sites; D019921:Biotinylation; D020118:Cupriavidus necator / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods*; D007700:Kinetics; D011091:Polyesters / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism; D019809:Streptavidin / Q000096:biosynthesis* / Q000235:genetics*
D000906:Antibodies; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D011091:Polyesters; D011993:Recombinant Fusion Proteins; D004247:DNA; D019809:Streptavidin; D000217:Acyltransferases; C060970:poly-beta-hydroxybutyrate polymerase