Abstract
Complex signaling cascades are difficult to study in vitro without phosphorylated proteins. Here, we describe a technique for the routine production of recombinant phosphoproteins by directly incorporating phosphoserine as a nonstandard amino acid. This protocol utilizes an optimized phosphoserine orthogonal translation system and an engineered strain of E. coli containing no genomic amber codons. This approach has been used to generate a variety of phosphorylated proteins to understand the role of protein phosphorylation in cell signaling.
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Acknowledgments
The authors would like to thank Natasha L. Pirman and Svetlana Rogulina for their efforts to establish this protocol. K.W.B. is supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1122492. J.R. is supported by NIH R01 GM117230 and NIDDK grant P01DK01743341.
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Barber, K.W., Rinehart, J. (2017). Expression of Recombinant Phosphoproteins for Signal Transduction Studies. In: Tan, AC., Huang, P. (eds) Kinase Signaling Networks. Methods in Molecular Biology, vol 1636. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7154-1_5
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DOI: https://doi.org/10.1007/978-1-4939-7154-1_5
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