title: A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems.
abstract: Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell-free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re-examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell-free system with a 95% reduction in reagent costs (excluding cell-extract, plasmid, and T7 RNA polymerase made in-house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze-thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high-throughput screening, and biotherapeutics.
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Qi
authors.initials: Q
authors.lastname: Cai
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Hanson
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Steiner
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Cuong
authors.initials: C
authors.lastname: Tran
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Mary Rose
authors.initials: MR
authors.lastname: Masikat
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Rishard
authors.initials: R
authors.lastname: Chen
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: James F
authors.initials: JF
authors.lastname: Zawada
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Aaron K
authors.initials: AK
authors.lastname: Sato
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Hallam
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, CA, 94080.
authors.forename: Gang
authors.initials: G
authors.lastname: Yin
mesh_terms: D003065:Coenzyme A / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D002955:Leucovorin / Q000737:chemistry; D009243:NAD / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011700:Putrescine / Q000737:chemistry; D019289:Pyruvic Acid / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry; D015203:Reproducibility of Results; D000068878:Trastuzumab / Q000096:biosynthesis* / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry
chemical_list: D007074:Immunoglobulin G; D011073:Polyamines; D014764:Viral Proteins; D009243:NAD; D018698:Glutamic Acid; D019289:Pyruvic Acid; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000068878:Trastuzumab; D002955:Leucovorin; D003065:Coenzyme A; D011700:Putrescine
keywords: IgG; cell-free; expression; immunoglobulin G; protein synthesis
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