2-Naphthylalanine → L-Tyrosine

HRID 605467Notes

Reaction equation

HRID 605467 reaction equation
C1C=[C:9]2[C:4]([CH:5]=[C:6]([CH2:11][C@H:12]([NH2:16])[C:13]([OH:15])=[O:14])[CH:7]=[CH:8]2)=CC=1.C([O-])(=O)CC[C@H](NC(C1C=CC(NCC2CNC3N=C(NC(C=3N=2)=O)N)=CC=1)=O)C(O)=[O:22]>>[NH2:16][C@H:12]([C:13]([OH:15])=[O:14])[CH2:11][C:6]1[CH:7]=[CH:8][C:9]([OH:22])=[CH:4][CH:5]=1

Participants

3

Reactant 2

HCID 1859152-Naphthylalanine2×HCID 136400727(4S)-4-[[4-[(2-amino-4-oxo-7,8-dihydro-3H-pteridin-6-yl)methylamino]benzoyl]amino]-5-hydroxy-5-oxopentanoate

Product 1

HCID 6057L-Tyrosine

Procedure

An L-3-(2-naphthyl)-alanine mutant of mouse dihydrofolate reductase (DHFR) was generated and characterized to confirm the ability of the mtRNACUATyr/SS12-TyrRS pair to site-specifically incorporate L-3-(2-naphthyl)-alanine in response to an amber stop codon. The Tyr163 codon of the mouse DHFR gene was mutated to TAG, and a His6 tag was added to the COOH-terminus of DHFR to facilitate protein purification using Ni2+ affinity chromatography. As a positive control, wild-type M. jannaschii TyrRS was coexpressed with the mtRNACUATyr resulting in efficient suppression of the TAG codon with tyrosine (FIG. 4). When SS12-TyrRS was coexpressed with the mu tRNACUATyr in the presence of 1 mM L-3-(2-naphthyl)-alanine, full-length mouse DHFR was also generated (with yield of 2.2 mg/L in liquid GMML minimal medium). In the absence of either L-3-(2-naphthyl)-alanine, mtRNACUATyr, or SS12-TyrRS, no full length DHFR was produced. A penta-His antibody was used to detect the His6 tag at the COOH-terminus of DHFR in a Western blot. No DHFR could be detected in the absence of each of the above three components.

Workup

  1. customprotein purification

Sources & evidence

ORD record
ord-dae77fc75a214c0992b6b8fc3183e99f
Source dataset
uspto-grants-2011_03
Patent number
US07915025B2

Reaction notes

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