title: A step-wise approach significantly enhances protein yield of a rationally-designed agonist antibody fragment in E. coli.
abstract: Fab59 is a rationally-designed antibody fragment (Fab) that mimics the activity of the cytokine thrombopoietin (TPO). Fab59 activity was initially detected directly from bacterial supernatants in a cell-based assay and was subsequently estimated to be equipotent to TPO using purified material. However, the expression of Fab59 was insufficient to support in vivo characterization of the Fab due to extremely low expression levels from its initial phage display expression vector. To boost expression, a new expression vector was designed and constructed, and Fab59 light chain codons were optimized for bacterial expression. However, from this a new challenge arose, in that the codon-optimized Fab59 was more toxic to Escherichia coli cells than parental Fab59. Co-expression of the bacterial chaperon protein Skp alleviated this toxicity. A two-step purification method was used to isolate monomeric Fab59 from the periplasm. Although Fab59 was prone to form aggregates during the purification process, buffer modification efficiently eliminated this problem. Overall, optimization of Fab59 expression and purification achieved a 100-fold increase in Fab59 production in E. coli relative to the starting yield. The yield of purified monomeric Fab59 from a shake flask reached up to 3.5mg/L, which was sufficient to support testing of the agonist activity of purified monomeric Fab59 in vivo. Even higher yields may be achieved by purification of Fab present in the culture media, as Skp most significantly increased accumulation of Fab59 in that location.
authors.affiliation: Alexion Antibody Technologies, Inc., 3985 Sorrento Valley Boulevard, Suite A, San Diego, CA 92121, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Lin
authors.forename: Mark W
authors.initials: MW
authors.lastname: Renshaw
authors.forename: Kathleen
authors.initials: K
authors.lastname: Autote
authors.forename: Lynette M
authors.initials: LM
authors.lastname: Smith
authors.forename: Peter
authors.initials: P
authors.lastname: Calveley
authors.forename: Katherine S
authors.initials: KS
authors.lastname: Bowdish
authors.forename: Shana
authors.initials: S
authors.lastname: Frederickson
mesh_terms: D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis; D018832:Molecular Chaperones / Q000096:biosynthesis; D018716:Molecular Mimicry; D010455:Peptides / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D053628:Receptors, Thrombopoietin / Q000819:agonists; D013926:Thrombopoietin / Q000276:immunology / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007140:Immunoglobulin Fab Fragments; D007147:Immunoglobulin Light Chains; D018832:Molecular Chaperones; D010455:Peptides; D053628:Receptors, Thrombopoietin; C515728:thrombopoietin mimetic peptide; C072721:Skp protein, E coli; D013926:Thrombopoietin