STyrRS | GCE4All Knowledge Base

RS/tRNA Foundational Publication Support

Liu, Chang C, and Peter G Schultz. (2006) 2006. “Recombinant Expression Of Selectively Sulfated Proteins In Escherichia Coli.”. Nature Biotechnology 24 (11): 1436-40.

Schwessinger, Benjamin, Xiang Li, Thomas L Ellinghaus, Leanne Jade G Chan, Tong Wei, Anna Joe, Nicholas Thomas, et al. (2016) 2016. “A Second-Generation Expression System For Tyrosine-Sulfated Proteins And Its Application In Crop Protection.”. Integrative Biology : Quantitative Biosciences From Nano To Macro 8 (4): 542-5. doi:10.1039/c5ib00232j.

Chen, Yuda, Shikai Jin, Mengxi Zhang, Yu Hu, Kuan-Lin Wu, Anna Chung, Shichao Wang, et al. (2022) 2022. “Unleashing The Potential Of Noncanonical Amino Acid Biosynthesis To Create Cells With Precision Tyrosine Sulfation.”. Nature Communications 13 (1): 5434. doi:10.1038/s41467-022-33111-4.

RS/tRNA Protocols and Structural Information

Liu, Chang C, Susan E Cellitti, Bernhard H Geierstanger, and Peter G Schultz. (2009) 2009. “Efficient Expression Of Tyrosine-Sulfated Proteins In E. Coli Using An Expanded Genetic Code.”. Nature Protocols 4 (12): 1784-9. doi:10.1038/nprot.2009.188.

RS/tRNA Usage Publications

Liu, Chang C, Antha Mack V, Meng-Lin Tsao, Jeremy H Mills, Hyun Soo Lee, Hyeryun Choe, Michael Farzan, Peter G Schultz, and Vaughn Smider V. (2008) 2008. “Protein Evolution With An Expanded Genetic Code.”. Proceedings Of The National Academy Of Sciences Of The United States Of America 105 (46): 17688-93. doi:10.1073/pnas.0809543105.

Liu, Chang C, Hyeryun Choe, Michael Farzan, Vaughn Smider V, and Peter G Schultz. (2009) 2009. “Mutagenesis And Evolution Of Sulfated Antibodies Using An Expanded Genetic Code.”. Biochemistry 48 (37): 8891-8. doi:10.1021/bi9011429.

RS/tRNA Pair Development Year

2006

ncAA(s) Incorporated

sulfo-L-tyrosine

ncAA Utility

Labelling of muscles (global)

Dipeptide (DanAla-‐Ala) required for uptake and bioavailability

RS Organism of Origin

Methanocaldococcus jannaschii

Parent RS

Tyr RS

RS Mutations

Y32L
L65P
D158G
I159C
L162K

tRNA Organism of Origin

Methanocaldococcus jannaschii

Parent tRNA

Tyr tRNA

tRNA Anticodon

CUA

Other tRNA Mutations

C17A
U17aG
U20C
G37A
U47G

RS/tRNA Availability

The RS is available in AddGene Plasmid #82417 (in the pULTRA plasmid system as shown in the second foundational publication)

The enzymes needed to make sulfoTyr endogenously in E. coli are encoded in Addgene #188983 (L-arabinose inducable)

Used in what cell line?

DH10B

C321.ΔA

BW25113 ΔcysH

RS/tRNA Additional Notes

In DH10B cells, with 2 mM sulfoTyr, gave high fidelity incorporation into Z-domain(7), noting that some (<10%) of the sulfo-group was lost during MS analysis. And with 10 mM sulfoTyr, yielded hirudin(63) at 5mg/L compared to WT yield of 12 mg/L. In the pULTRA plasmid yielded ~75% efficient expression of GFP with one TAG site and ~25% efficient expression of GFP with three TAG sites, and high fidelity using the RF1- C321.ΔA cell line.

The Chen et al. 2022 foundational paper reports how sulfo-Tyr can also be biosynthesized using the plasmid pEvol-NnSULT1C1-cysDNCQ (Addgene #188983) in the BW25113 ΔcysH E. coli strain, providing ~10-fold improvement in sfGFP-TAG expression compared to supplementing media with 1 mM sTyr.