| Popis: |
This protocol describes the recombinant expression of proteins in E. coli containing phosphoserine (pSer) installed at positions guided by TAG codons. The E. coli strains that can be used here are engineered with a ∆serB genomic knockout to produce pSer internally at high levels, so no exogenously added pSer is required, and the addition of pSer to the media will not affect expression yields. For “truncation-free” expression and improved yields with high flexibility of construct design, it is preferred to use the Release Factor-1 (RF1) deficient strain B95(DE3) ∆A ∆fabR ∆serB, though use of the standard RF1-containing BL21(DE3) ∆serB is also described. Both of these strains are serine auxotrophs and will not grow in standard minimal media. This protocol uses rich auto-induction media for streamlined and maximal production of homogeneously modified protein, yielding ~100–200 mg of single pSer-containing sfGFP per liter of culture. Using this genetic code expansion (GCE) approach, in which pSer is installed into proteins during translation, allows researchers to produce milligram quantities of specific phospho-proteins without requiring kinases, which can be purified for downstream in vitro studies relating to phosphorylation-dependent signaling systems, protein regulation by phosphorylation, and protein–protein interactions.Graphical abstract: |
