Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimen-tally ascertain their effects on protein structure and function. Therefore, the production of 15N-(and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site -specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic Delta serB muta-tion, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) Delta serB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimi-zation and adaption of a high-density culture protocol, we were able to obtain >= 10 mg/L homogenously labeled, phosphory-lated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/ arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We pro-pose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.

Automated summary

Phosphoserine (pSer) sites are primarily located within disordered protein regions. Producing labeled proteins with site-specific pSer for NMR studies is important for understanding the molecular mechanisms of protein regulation. However, methodologies for adapting pSer genetic code expansion to minimal or isotope-enriched media have not been described.