High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids.
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).
Goerke
Aaron R
AR
Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
Swartz
James R
JR
D000596:Amino Acids / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D017017:Methanococcus / Q000201:enzymology; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D014176:Protein Biosynthesis; D012365:RNA, Transfer, Tyr / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D001386:Azides; D012365:RNA, Transfer, Tyr; C027162:4-azidophenylalanine; D010649:Phenylalanine; D014448:Tyrosine-tRNA Ligase