title: A designed, phase changing RTX-based peptide for efficient bioseparations.
abstract: Typically, chromatography is the most costly and time-consuming step in protein purification. As a result, alternative methods have been sought for bioseparations, including the use of stimulus-responsive tags that can reversibly precipitate out of solution in response to the appropriate stimulus. While effective, stimulus-responsive tags tend to require temperature changes or relatively harsh buffer conditions to induce precipitation. Here we describe a synthetic peptide, based on the natural repeat-in-toxin (RTX) domain that undergoes gentler calcium-responsive, reversible precipitation. When coupled to the maltose binding protein (MBP), our calcium-responsive tag efficiently purified the fusion protein. Furthermore, when the MBP was appended to green fluorescent protein (GFP), β-lactamase, or a thermostable alcohol dehydrogenase (AdhD), these constructs could also be purified by calcium-induced precipitation. Finally, protease cleavage of the precipitating tag enables the recovery of pure and active target protein by cycling precipitation before and after cleavage.
authors.affiliation: Department of Chemical Engineering, Columbia University, New York, NY, USA.
authors.forename: Oren
authors.initials: O
authors.lastname: Shur
authors.forename: Kevin
authors.initials: K
authors.lastname: Dooley
authors.forename: Mark
authors.initials: M
authors.lastname: Blenner
authors.forename: Matthew
authors.initials: M
authors.lastname: Baltimore
authors.forename: Scott
authors.initials: S
authors.lastname: Banta
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001886:Bordetella pertussis / Q000737:chemistry* / Q000235:genetics; D002118:Calcium / Q000737:chemistry*; D011232:Chemical Precipitation; D003001:Cloning, Molecular / Q000379:methods; D016384:Consensus Sequence; D006801:Humans; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D037361:Adenylate Cyclase Toxin; D058619:Maltose-Binding Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases; D002118:Calcium