title: Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli.
abstract: Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mgL(-1) culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships.
authors.affiliation: Pacific Northwest National Laboratory, 902 Battelle Boulevard, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Miller
authors.forename: Jane
authors.initials: J
authors.lastname: Weaver-Feldhaus
authors.forename: Sean A
authors.initials: SA
authors.lastname: Gray
authors.forename: Robert W
authors.initials: RW
authors.lastname: Siegel
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Feldhaus
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins