High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids |
| |
Authors: | Goerke Aaron R Swartz James R |
| |
Affiliation: | Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA. |
| |
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 microg/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 microg/mL of eCAT109pAz, 660 microg/mL of eDHFR10pAz, and 210 microg/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). |
| |
Keywords: | non‐natural amino acid cell‐free protein synthesis click chemistry E. coli inner membrane vesicles Methanococcus jannaschii unnatural amino acid (3 + 2) cycloaddition reaction |
本文献已被 PubMed 等数据库收录! |
|