High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids
✍ Scribed by Aaron R. Goerke; James R. Swartz
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 500 KB
- Volume
- 102
- Category
- Article
- ISSN
- 0006-3592
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
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 tRNA^Tyr^ pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200–300 µg/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 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/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). Biotechnol. Bioeng. 2009;102: 400–416. © 2008 Wiley Periodicals, Inc.