Separation and Purification of Recombinant ß-Glucosidase with Hydrophobicity and Thermally Responsive Property from Cell Lysis Solution by Foam Separation and Further Purification.

The aim of this study was to separate and purify recombinant ß-glucosidase (GLEGB) with elastin-like polypeptide (ELP) and graphene-binding peptide (GB) from cell lysis solution by foam separation and further purification. The study of foam property of GLEGB cell lysis solution indicated that it had excellent foaming property and foam stability, which was suitable for foam separation. This could be due to the GB tag with hydrophobicity, which made the recombinant ß-glucosidase with GB preferentially adsorb on the surface of bubbles. At optimum operating conditions of foam separation, the enzyme activity recovery of GLEGB could reach 95.63 ± 1.0%. The foam solution of GLEGB was further purified based on the thermally responsive property of the ELP tag, and the purification fold of GLEGB could reach 29.6 ± 0.5 at the optimum operating conditions. The prominent purification effect indicates that this technique is a simple and efficient technique for the separation and purification of recombinant enzymes.

Combination of aqueous two-phase flotation and inverse transition cycling: Strategies for separation and purification of recombinant ß-glucosidase from cell lysis solution.

This work was developed to solve the problems of the restriction of non-specific adsorption and time-dependent denaturation in the purification of recombinant proteins by multistage chromatographic procedures. A novel purification method (ATPF-ITC) which combining aqueous two-phase flotation (ATPF) with inverse transition cycling (ITC) was established and used to efficiently purify recombinant ß-glucosidase (GLEGB) from cell lysis solution. First, GLEGB would preferentially adsorb on the nitrogen bubble interface relied on the hydrophobic property of the graphene-binding (GB) tag and enter into the top phase of ATPF. Second, GLEGB was achieved further purification by one-round ITC method based on the thermosensitive of the elastin-like polypeptide (ELP) tag. Consequently, the enzymatic activity recovery of GLEGB was 124.92% ± 0.83%, and the purification factor reached 24.26 ± 0.22. The purification results remained stable after six polymer cycles, and the process of ATPF-ITC had no negative effect on the structure of recombinant protein.