title: Development of cell-free protein synthesis platforms for disulfide bonded proteins.
abstract: The use of cell-free protein synthesis (CFPS) for recombinant protein production is emerging as an important technology. For example, the openness of the cell-free system allows control of the reaction environment to promote folding of disulfide bonded proteins in a rapid and economically feasible format. These advantages make cell-free protein expression systems particularly well suited for producing patient specific therapeutic vaccines or antidotes in response to threats from natural and man-made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. In this work we assess the versatility of modern cell-free methods, optimize expression and folding parameters, and highlight the importance of rationally designed plasmid templates for producing mammalian secreted proteins, fusion proteins, and antibody fragments in our E. coli-based CFPS system. Two unique CFPS platforms were established by developing standardized extract preparation protocols and generic cell-free reaction conditions. Generic reaction conditions enabled all proteins to express well with the best therapeutic protein yield at 710 microg/mL, an antibody fragment at 230 microg/mL, and a vaccine fusion protein at 300 microg/mL; with the majority correctly folded. Better yields were obtained when cell-free reaction conditions were optimized for each protein. Establishing general CFPS platforms enhances the potential for cell-free protein synthesis to reliably produce complex protein products at low production and capital costs with very rapid process development timelines.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Goerke
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D000818:Animals; D002474:Cell-Free System; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D051379:Mice; D014176:Protein Biosynthesis; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014612:Vaccines / Q000096:biosynthesis
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D014612:Vaccines