• These protocols have been tested by the GCE4All Research Center and resources needed for the protocols are generally available through Addgene.
• These protocols are reliable and robust for expressing the sfGFP florescence reporter protein with the relevant non-canonical amino acid (ncAA) incorporated at position 150 (sfGFP-150-ncAA).
• Confirming sfGFP-150-ncAA expression at near stated yields is recommended as the first step for anyone seeking to use the protocol; then sfGFP-150-ncAA expression can always be used as a control for the process.
• Every protein of interest (POI) to be expressed will have unique characteristics, and thus optimized expression of a given POI may require modifications of the protocol.
• For help with protocol implementation and troubleshooting, as well as experimental design, please join the GCE bulletin Board list-serve and post your questions/requests there to access support from members of the GCE4All Research Center and the broader GCE community.

GCE4All-1: For expression in E. coli of proteins containing phosphoserine

Citation: Zhu P, Mehl RA, Cooley RB (2022) Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli. Bio-protocol 12:e4541. doi: 10.21769/BioProtoc.45.

Notes:

A method is also published for using this GCE system to make ¹³C-, ¹⁵N-isotopically labeled proteins containing phosphoserine such as would be useful for NMR studies. The expression methods for isotopic labeling of pSer proteins are different than for standard expressions because the ∆serB expression hosts are serine auxotrophs and therefore cannot grow in conventional minimal media.

• The basic method is in Vesely et al. (2022) Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions. J Biol Chem. 298:102613. doi: 10.1016/j.jbc.2022.102613
• An adaption of the isotopic labeling protocol for the expression of a particular POI is in Buchko et al. (2023) High-yield recombinant bacterial expression of ¹³C-, ¹⁵N-labeled, serine-16 phosphorylated, murine amelogenin using a modified third generation genetic code expansion protocol. Protein Sci32:e4560. doi: 10.1002/pro.4560

GCE4All-2: For expression in E. coli of proteins containing non-hydrolyzable phosphoserine (the “Permaphos” system)

Citation: Zhu P, Mehl RA, Cooley RB (2023) Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia Coli. Bio-protocol 13: e4861. DOI: 10.21769/BioProtoc.4861.

GCE4All-3: Tetrazine Amino Acid Encoding for Rapid and Complete Protein Bioconjugation

Citation: Eddins, A. J., Pung, A. H., Cooley, R. B. and Mehl, R. A. (2024). Tetrazine Amino Acid Encoding for Rapid and Complete Protein Bioconjugation. Bio-protocol 14(16): e5048. DOI:10.21769/BioProtoc.5048