About our protocols

• 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).
• Many GCE4All Protocols are published in partnership with bio-protocol, an online peer-reviewed protocol journal curating and hosting high quality, free access, step-by-step protocols across the life sciences.

Using these protocols

• 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 Protocol

For expression in E. coli of proteins containing phosphoserine

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.

Additional resources

• The B95(DE3) ∆A ∆fabR ∆serB strain is available from Addgene
• Webinar Recording: "PermaPhos:Revealing New Functions of Phosphorylated Proteins"

To make ¹³C-, ¹⁵N-isotopically labeled proteins containing phosphoserine (for NMR studies)

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

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.

Additional resources

• Adaption of the isotopic labeling protocol for the expression of a particular POI: 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
• Webinar Recording: "NMR isotope-labelled, serine-16 phosphorylated, amelogenin"

GCE4All-2 Protocol

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

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.

Additional resources

• Components for this work are available as a prepackaged PermaPhos kit on Addgene
• Webinar Recording: "PermaPhos:Revealing New Functions of Phosphorylated Proteins"

GCE4All-3 Protocol

Tetrazine Amino Acid Encoding for Rapid and Complete Protein Bioconjugation

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

Additional resources

• The pAJE3-E7 plasmid is available on Addgene

GCE4All-4

Selecting aminoacyl-tRNA synthetase/tRNA pairs for efficient genetic encoding of noncanonical amino acids into proteins

Alexander N. D., Gangarde Y. M., Bednar R. M., Karplus P. A., Cooley R. B. and Mehl R. A. (2025). Selecting aminoacyl-tRNA synthetase/tRNA pairs for efficient genetic encoding of noncanonical amino acids into proteins. Nat Protoc DOI: 10.1038/s41596-025-01241-w.

Additional resources

• Selection plasmids are available on Addgene