FIGURE 2.

Polysaccharide‐binding activity of His‐tagged PHEC27213. (a) In vitro expression and purification of His‐tagged PHEC27213 protein. This protein was expressed in the Escherichia coli Bl21‐CodonPlus‐RIL and purified using Protino Ni‐TED 2000 Packed Columns (Macherey‐Nagel GmbH & Co). The image shows the different steps of protein purification visualized using Mini‐PROTEAN Stain‐Free Precast Gels (Bio‐Rad) in a ChemiDoc XRS + system (Bio‐Rad). The arrow indicates the band corresponding to the soluble purified protein. Lanes are MW (protein marker), Precision Plus Protein Unstained Standard (Bio‐Rad); P, pellet sample; L, supernatant sample after cell lysing; FT, discarded flow‐through sample after protein‐column binding; W1, first wash sample; W2, second wash sample; E, eluted soluble protein sample. (b) Polysaccharide‐binding activity of His‐tagged PHEC27213 protein. Quantification of soluble proteins (supernatant fraction) after incubation of soluble PHEC27213 (1 mg/ml) for 60 min with colloidal chitin or cellulose (10 mg/ml). Bovine serum albumen (BSA) was used as a negative control. Bars indicate the standard error of three technical replicates from three different experiments. (c) Western blot analysis of the His‐tagged PHEC27213 protein from the supernatant samples of the polysaccharide‐binding assay described in (b). The lanes are MW (protein marker), Precision Plus Protein Unstained Standard (Bio‐Rad); E (eluted protein), soluble PHEC27213 (1 mg/ml) taken from the final elution step after protein expression; P (pellet fraction), presence of PHEC27213 in the pellet after incubation with colloidal chitin or cellulose; Ch (chitin), colloidal chitin solution; Ce (cellulose), cellulose solution; S (supernatant fraction), presence of PHEC27213 in the supernatant after incubation with colloidal chitin or cellulose. Arrows indicate the bands corresponding to the PHEC27213 protein