PXD027053

PXD027053 is an original dataset announced via ProteomeXchange.

Title	Characterization of two mutually orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pairs residing in the same organism
Description	Site-specific incorporation of distinct non-canonical amino acids (ncAAs) into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. Pyrrolysyl–tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel PylRS/tRNAPyl pairs from extremely halophilic methyl-reducing euryarchaeal HMET1. In order to demonstrate that HMET1 PylRS/tRNAPyl pairs are functional and orthogonal in H. volcanii, we transformed plasmids encoding HMET1 PylRS/tRNAPyl pair and reporter protein SAMP1(G24amb), and cultured the cells in the presence and absence of 1 mM ncAA. Amber suppression ability of HMET1 PylRS/tRNAPyl pairs were assayed by monitoring the production of the full-length SAMP1(G24amb) and the SAMP1(G24amb)-MoaE conjugate via anti-Flag immunoblotting. LC-MS/MS confirmed that BocK (ncAA) was incorporated into the reporter protein at site-directed position. Thus, these two distinct PylRS/tRNAPyl pairs are functional in Haloferax volcanii, a model halophilic archaeon. Furthermore, by swapping these two PylRS/tRNAPyl pairs in the expression vector followed by anti-Flag immunoblotting, we found these two PylRS/tRNAPyl pairs are mutually orthogonal. Finally, we demonstrate HMET1 PylRS/tRNAPyl-derived pairs can decode two stop codons and incorporate distinct ncAAs. LC-MS/MS analysis of the purified reporter protein confirmed that BocK and 3-I-phe were incorporated at the TAA and TAG-directed positions respectively.
HostingRepository	PRIDE
AnnounceDate	2022-05-12
AnnouncementXML	Submission_2022-05-12_02:12:50.898.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Yuxing Zhang
SpeciesList	scientific name: Haloferax volcanii; NCBI TaxID: 2246;
ModificationList	monohydroxylated residue; iodoacetic acid derivatized residue; deamidated residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2021-07-01 23:30:09	ID requested
⏵ 1	2022-05-12 02:12:51	announced

Zhang H, Gong X, Zhao Q, Mukai T, Vargas-Rodriguez O, Zhang H, Zhang Y, Wassel P, Amikura K, Maupin-Furlow J, Ren Y, Xu X, Wolf YI, Makarova KS, Koonin EV, Shen Y, S, ö, ll D, Fu X, The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism. Nucleic Acids Res, 50(8):4601-4615(2022) [pubmed]

submitter keyword: pyrrolysyl-tRNA synthetase, tRNAPyl, archaea, genetic code expansion, mutually orthogonal

Xian Fu
contact affiliation	BGI-Shenzhen, Shenzhen, 518083,China
contact email	Fuxian1@genomics.cn
lab head
Yuxing Zhang
contact affiliation	Building 11, Beishan Industrial Zone, Yantian District, Shenzhen (518083)（518083）
contact email	745682036@qq.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD027053
     1. Label: PRIDE project
     2. Name: Characterization of two mutually orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pairs residing in the same organism