A novel approach for purification of recombinant proteins using the dextran‐binding domain

Abstract

Using the dextran-binding domain (DBD) of a type of glucosyltransferase (GTF) from Streptococcus sobrinus, we have developed a novel method for purifying recombinant proteins. DBD-tagged green and red fluorescent proteins as well as the parent GTF and DBD moiety were adsorbed well to commercially available cross-linked dextran (such as Sephadex beads and Sephacryl beads), and eluted efficiently with water-soluble dextran. The purity of the eluted proteins after this one-step affinity purification was V V90% or better. The results suggest that DBD can be used as a powerful carrier for purification of various recombinant proteins. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Search for a dextransucrase minimal motif involved in dextran binding

“…DGTIVYFDKK-GHQVFDQYIT YG 4 NGNAYYFDDA-GVMLKSGLATI YG 5 DGHQQYFDQN-GVQVKDKFVIG YG 6 -NQWFYFDGN-GHAVT-GFQTI YG 7 NGKKQYFYND-GHQSK YG 8 -DGDTFYTSATDGRLVT-GVQKI YG 9 NGITYAFDNT-GNLI YG 10 TN-QYYQLAD-GKYMLLDDSGR YG 11 DGVLRYFDQN-GEQVKDAIIVD YG 12 DTNLSYYFNATQGVAV YG 13 -KNDYFEYQ-GNWY YG 14 TD-ANYQLIK-GFKAVDD YG 15 AQGKVYQFDNN-GNAVSA Boldface type indicates conserved amino acids, x corresponds to nonconserved amino acids, and H°to hydrophobic residues. ary and tertiary structure formation.…”

“…However, our tag is around half the size of theirs and is also shorter than commercial tags such as GST or MBP. In addition, the protein elutions can be performed using a smaller molecule (1.5 kDa dextran instead of 18 kDa) at a lower concentration (5 g/l of competitor compared to 50 g/l in Kaseda et al [9] procedure). Moreover the potential for gentle elution by a small and neutral molecule like isomaltohexaose will limit enzyme inhibition or denaturation.…”

“…For example, the strong affinity for dextran has been used to purify glucansucrases by dextran/polyethyleneglycol phase demixion [5], or affinity chromatography onto Sephadex Ò gels [6][7][8], which consist of crosslinked dextrans. Kaseda et al [9] proposed the use of the 30 kDa GBD of Streptococcus sobrinus GTF-I glucansucrase as an affinity tag for recombinant protein purification. More recently, Shah et al [10] revealed the crucial role of the aromatic residues found in the YG repeats, which were suggested to ''stack'' with the sugar units of dextran or mutan.…”

Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system

“…As described above, GTF in the CCC fractions is readily recovered from the PTFE column whereas the strong affinity of GTF to dextran is a severe drawback in gel-filtration chromatography using Sephadex beads as a solid support [19]. However, these CCC fractions contain high concentrations of high dextran T500 which cannot be easily removed by dialysis or ultrafiltration.…”

“…However, the recovery of GTF was not satisfactory in these methods due to the irreversible adsorption of GTF to the column packing materials. In particular, GTFs have some dextran-binding domains leading to the irreversible adsorption of cross-linked dextran such as Sephadex and Sephacryl beads [19]. Therefore, it is desirable to find an alternative method which isolates GTF at a high yield without loss of its enzymatic activity.…”