A Highly Versatile Expression System for the Production of Multiply Phosphorylated Proteins.

Genetic Code Expansion (GCE) can use TAG stop codons to guide site-specific incorporation of phosphoserine (pSer) into proteins. To eliminate prematurely truncated peptides, improve yields, and enhance the production of multiphosphorylated proteins, Release Factor 1 (RF1)-deficient expression hosts were developed, yet these grew slowly and their use was associated with extensive misincorporation of natural amino acids instead of pSer. Here, we merge a healthy RF1-deficient E. coli cell line with a high-efficiency pSer GCE translation system to produce a versatile pSer GCE platform in which only trace misincorporation of natural amino acids is detected even when five phosphoserines were introduced into one protein. Approximately 400 and 200 mg of singly and doubly phosphorylated GFP per liter of culture were obtained. Importantly, the lack of truncated protein permits expression of oligomeric proteins and the use of N-terminal solubility-enhancing proteins to aid phospho-protein expression and purification. To illustrate the enhanced utility of this system, we produce doubly phosphorylated STING (Stimulator of Interferon Genes), as well as triply phosphorylated BAD (Bcl2-associated agonist of cell death) complexed with 14-3-3, in quantity, purity, and homogeneity sufficient for structural biology applications. We anticipate that the facile access to phosphoproteins enabled by this system, which we call pSer-3.1G, will expand studies of the phospho-proteome.