title: When less becomes more: optimization of protein expression in HEK293-EBNA1 cells using plasmid titration - a case study for NLRs.
abstract: Transient transfection of the human HEK293-EBNA1 cell line using polyethyleneimine is widely adopted for recombinant protein production. Whereas high expression of many targets is achieved, purification yields of some highly expressed proteins remain low due to aggregation. We hypothesized that for these proteins the expression rates achieved at standard transfection conditions are too high, causing an overload of the protein folding machinery. Here we present plasmid titration as an efficient method to vary expression rates for the optimization of soluble protein expression. In plasmid titration a dilution series of expression vector mixed with dummy plasmid is transfected in small scale cultures. Application to GFP shows that plasmid titration achieves a wide range of expression levels while maintaining high transfection efficiencies even at 500-fold plasmid dilution. Application of plasmid titration to selected Nod-like receptors (NLRs), which at standard conditions are highly expressed but poorly soluble, delays the onset of NLR aggregation and improves cell viability and the buildup of biomass. The amount of soluble protein depends on the combination of dilution factor and harvest day in a protein specific manner. For NOD1 50-fold plasmid dilution increases the amount of soluble protein approximately 5-fold. Due to its association with chaperones at all dilution factors tested we were unable to purify NOD1 to homogeneity. For NLRC4, which did not associate with chaperones, 10-fold plasmid dilution increased the purification yield 2-fold. This improvement, obtained with minimal effort due to the simplicity of the method, shows that reducing total expression may increase soluble protein yield.
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Els F
authors.initials: EF
authors.lastname: Halff
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Marian
authors.initials: M
authors.lastname: Versteeg
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: T Harma C
authors.initials: TH
authors.lastname: Brondijk
authors.affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands. Electronic address: e.g.huizinga@uu.nl.
authors.forename: Eric G
authors.initials: EG
authors.lastname: Huizinga
mesh_terms: D053468:CARD Signaling Adaptor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016923:Cell Death; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells / Q000378:metabolism*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011094:Polyethyleneimine; D066329:Protein Aggregates / Q000502:physiology*; D017510:Protein Folding / Q000187:drug effects; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics; D012995:Solubility; D014162:Transfection / Q000379:methods*
chemical_list: D053468:CARD Signaling Adaptor Proteins; D002135:Calcium-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; C435692:NLRC4 protein, human; D066329:Protein Aggregates; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; C417029:EBV-encoded nuclear antigen 1
keywords: HEK293E cells; Nod-like receptors; Protein aggregation; Recombinant protein expression; Transient transfection