title: Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.
abstract: In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and β-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.
authors.affiliation: Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA.
authors.forename: Wan-Yi
authors.initials: WY
authors.lastname: Wu
authors.forename: Keith D
authors.initials: KD
authors.lastname: Miller
authors.forename: Michael
authors.initials: M
authors.lastname: Coolbaugh
authors.forename: David W
authors.initials: DW
authors.lastname: Wood
mesh_terms: D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D047668:Inteins; D009169:Mycobacterium tuberculosis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D004220:Disulfides; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins; D002686:Chitin