title: Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage.
abstract: While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale-up have promoted the development of alternative non-chromatographic separation methods. Here we developed a new non-chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo-responsive elastin-like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin-dockerin pairs, which are high-affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Shen-Long
authors.initials: SL
authors.lastname: Tsai
authors.forename: Bhawna
authors.initials: B
authors.lastname: Madan
authors.forename: Wilfred
authors.initials: W
authors.lastname: Chen
mesh_terms: D001709:Biotechnology / Q000379:methods*; D011232:Chemical Precipitation; D048013:Acetivibrio thermocellus / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D047668:Inteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins