期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 102, 期 2, 页码 400-416出版社
WILEY
DOI: 10.1002/bit.22070
关键词
non-natural amino acid; cell-free protein synthesis; click chemistry; E. coli inner membrane vesicles; Methanococcus jannaschii; unnatural amino acid; (3+2) cycloaddition reaction
资金
- Merck Co., Inc.
- Merck BioPurification Department
We describe an E. coli-based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200-300 mu g/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m(2)/mL This new E. coli-based cell-free procedure produced up to 400 mu g/mL, of eCAT109pAz, 660 mu g/mL of eDHFR10pAz, and 210 mu g/mL of mDHFR31pAz with p-azido-L-phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O-methyl-L-tyrosine and p-acetyl-L-phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper (I)-catalyzed (3 + 2) cycloadditions (click chemistry).
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