Combination of aqueous two-phase flotation and inverse transition cycling: Strategies for separation and purification of recombinant β-glucosidase from cell lysis solution. (30th March 2022)

• Record Type: Journal Article
• Authors: Han, Juan; Fang, Sihan; He, Xingchen; Wang, Lei; Li, Chunmei; Wu, Jiacong; Cai, Yunfeng; Wang, Yun
• Is Part Of: Food chemistry. Volume 373:Part B(2022)
• Publication Date: 2022-03-30
• Subjects: Separation and purification -- Recombinant β-glucosidase -- Flotation -- Elastin-like polypeptide (ELP) -- Graphene-binding peptide (GB); Food -- Analysis -- Periodicals; Food -- Composition -- Periodicals; 664
• DOI: 10.1016/j.foodchem.2021.131543
• Languages: English
• ISSNs: 0308-8146

Abstract

Highlights: A recombinant protein was designed to incorporate a GB and ELP tags based on Glu. A new purification method which combining ATPF system with ITC was established. GLEGB was achieved preliminary purification by ATPF relied on GB tag. The further purification of GLEGB was achieved by one-round ITC based on the ELP tag. The purification efficiency was satisfactory with the purification factor reached 24.26 ± 0.22. Abstract: 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 β-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.