Identification of peptides specific for dextran binding by glucan binding assay technique

Abstract

One of the key steps in protein characterization and functional analysis is protein purification. Fusion of an affinity tag to protein of interest is enabling the protein separation from other contaminants in the extract through an affinity chromatography step. Considering a C-terminal domain of dextransucrase from Leuconostoc mesenteroides NRRL B-512F (DSR-S), it often refers as a glucan-binding domain (GBD). However. Amino acid sequences responsible for the glucan binding have not been totally elucidate yet. The objectives of our study are to investigate the precise role of GBD in glucan binding and to generate shortest fragment that could be employed as a tag for affinity purification onto available cross-linked dextran supports. Fourteen truncated forms of DSR-S C-terminal domain were tested for their ability to bind dextrans. With a 14 kDa molecular weight, the shortest fragment (GBD-7) exhibiting a strong interaction with dextran was found to be localized at C-terminal end of the GBD and consists of six YG repeats. With a dissociation constant Kd of 2.8 10-9 M, this motif shows a very high affinity for isomaltohexaose and longer dextrans. Confirming a very strong dextran binding property. Potential application of GBD0-7 to be used as an affinigy tag for rapid purification onto purity higher than 95% after competitive elution using 1.5 kDa dextran. Moreover. Rare codons representing in the GBD0-7 sequences were replaced by frequently-used codons in E. coli by site-directed mutagenesis. This approach cold enhance the fusion protein expression up to 4.8 folds of a wild-type. In order to increase its solubility and simultaneously reduce its size. nested truncated forms of GBD-7 proteins were generated. From the GBD-7 sequences. it can be deleted only 12 amino acids at N-terminus with retained binding activity. The truncations at C-terminal V5 epitope part of the GBD-7 fusion protein could enhance its expression in with or without Thioredoxin tag.