title: High-throughput purification of recombinant proteins using self-cleaving intein tags.
abstract: High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.
authors: Process Development, Sanofi-Genzyme, 31 New York Ave, Framingham, MA 01701, USA.
authors: M J
authors: MJ
authors: Coolbaugh
authors: Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA.
authors: M J
authors: MJ
authors: Shakalli Tang
authors: Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA. Electronic address: wood.750@osu.edu.
authors: D W
authors: DW
authors: Wood
mesh_terms: D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D047668:Inteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase
keywords: Affinity methods; Elastin-like polypeptide; High throughput protein purification; Intein tag; Recombinant protein purification; Self-cleaving tag