Improved adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding domain from Aspergillus glucoamylase.
We have previously shown (Chen et al., 1991) that a beta-galactosidase (beta-gal) fusion protein (BSB133) containing 133 amino acids (aa) from the C-terminus of Aspergillus glucoamylase (GA) adsorbs strongly to starch compared to beta-gal, due to the presence of the GA starch-binding domain. We have now made deletions at the N-terminus of this 133-aa region to test the minimal size required for starch binding of beta-gal fusion proteins. Three fusion proteins (BSB119, BSB103, and BSB80) were genetically engineered, containing 119, 103, and 80 C-terminal aa from GA, respectively. The fusion proteins were expressed in Escherichia coli and purified. Purified BSB119 adsorbed to native starch at least 2-fold more strongly than did BSB133 or fusion proteins with shorter tails. Adsorption isotherms generated over a wide range of initial concentrations indicated a 10-fold difference in the loading capacity of starch for BSB119 (36.5 mg of protein/g of starch) compared to beta-gal (3.7 mg of protein/g of starch). Adsorption constants calculated from the initial slopes of the isotherms indicated a nearly 30-fold difference in affinity to starch for BSB119 (Kad = 63 mL/g of starch) compared to beta-gal (Kad = 2.3 mL/g of starch). BSB119 in the presence of crude enzyme extracts also bound to starch with a high affinity compared to a beta-gal control. Potential applications of the starch-binding tail include enzyme immobilization to starch or recovery and purification of target proteins from crude extracts.
Chen
L J
LJ
Department of Genetics, Iowa State University, Ames 50011.
Ford
C
C
Kusnadi
A
A
Nikolov
Z L
ZL
D000327:Adsorption; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013213:Starch / Q000737:chemistry*; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D013213:Starch; D001616:beta-Galactosidase; D005087:Glucan 1,4-alpha-Glucosidase