Adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding region of Aspergillus glucoamylase.
We have constructed and purified by affinity chromatography three beta-galactosidase (beta Gal) fusion proteins (BSB133, BSBCD8, and BGA134) containing amino acid (aa) sequences from Aspergillus glucoamylase (GA). BSB133, containing the C-terminal 133 aa of GA (aa 484-616), adhered to native starch granules with a much higher affinity (Kad = 18 ml/g starch) than a beta Gal control (Kad = 0.9 ml/g starch). Two other fusion proteins, BSBCD8 and BGA134, similar in size to BSB133, adhered to starch with a relatively low affinity (Kad = 7 ml/g starch, and Kad = 4 ml/g starch, respectively). BSBCD8 differs from BSB133 by a truncation of 8 aa at the C terminus. BGA134 contains 134 aa from an overlapping region of GA (aa 380-513). These results confirm the presence of a strong starch-binding region (SBR) included in the C-terminal 133 aa of GA and indicate that the SBR can confer starch-binding activity on a fusion protein produced in Escherichia coli. In the presence of crude soluble cell extracts, the fusion proteins adsorbed by native starch granules with an affinity similar to that of the purified enzymes. BSB133 that had been adsorbed by starch from crude extracts could be eluted at a high level of purity, similar to that achieved by affinity chromatography. These results suggest that it may be feasible to use native starch as an adsorbent for the recovery and purification of recombinant fusion proteins containing the SBR. Starch has many favorable qualities for this application: it is inexpensive, stable, nontoxic, and easy to recover by centrifugation.
Chen
L J
LJ
Department of Genetics, Iowa State University, Ames 50011.
Ford
C
C
Nikolov
Z
Z
D000327:Adsorption; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013213:Starch / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013213:Starch; D001616:beta-Galactosidase; D005087:Glucan 1,4-alpha-Glucosidase