کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020451 1542342 2014 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
When less becomes more: Optimization of protein expression in HEK293–EBNA1 cells using plasmid titration – A case study for NLRs
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
When less becomes more: Optimization of protein expression in HEK293–EBNA1 cells using plasmid titration – A case study for NLRs
چکیده انگلیسی


• A subset of proteins aggregate upon transient expression in HEK293E cells.
• This may be caused by the high expression levels achieved by this system.
• Plasmid titration is a rapid and straightforward method to vary expression levels.
• Lowering expression levels reduces total, but increases soluble expression of NLRs.

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 99, July 2014, Pages 27–34
نویسندگان
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