Abstract
The genetic incorporation of unnatural amino acids (UAAs) into proteins has been a useful tool for protein engineering. However, most UAAs are expensive, and the method requires a high concentration of UAAs, which has been a drawback of the technology, especially for large-scale applications. To address this problem, a method to recycle cultured UAAs was developed. The method is based on recycling a culture medium containing the UAA, in which some of essential nutrients were resupplemented after each culture cycle, and induction of protein expression was controlled with glucose. Under optimal conditions, five UAAs were recycled for up to seven rounds of expression without a decrease in expression level, cell density, or incorporation fidelity. This method can generally be applied to other UAAs; therefore, it is useful for reducing the cost of UAAs for genetic incorporation and helpful for expanding the use of the technology to industrial applications.
This is a preview of subscription content, log in via an institution to check access.
References
Chatterjee A, Guo J, Lee HS, Schultz PG (2013) A genetically encoded fluorescent probe in mammalian cells. J Am Chem Soc 135:12540–12543
Chin JW (2014) Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem 83:379–408
Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG (2002) Addition of p-azido-l-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc 124:9026–9027
Drienovska I, Rioz-Martinez A, Draksharapu A, Roelfes G (2015) Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids. Chem Sci 6:770–776
Grossman TH, Kawasaki ES, Punreddy SR, Osburne MS (1998) Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability. Gene 209:95–103
Jung JE, Lee SY, Park H, Cha H, Ko W, Sachin K, Kim DW, Chi DY, Lee HS (2014) Genetic incorporation of unnatural amino acids biosynthesized from α-keto acids by an aminotransferase. Chem Sci 5:1881–1885
Lang K, Davis L, Torres-Kolbus J, Chou C, Deiters A, Chin JW (2012a) Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat Chem 4:298–304
Lang K, Davis L, Wallace S, Mahesh M, Cox DJ, Blackman ML, Fox JM, Chin JW (2012b) Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic diels–alder reactions. J Am Chem Soc 134:10317–10320
Lee HS, Schultz PG (2008) Biosynthesis of a site-specific DNA cleaving protein. J Am Chem Soc 130:13194–13195
Lee HS, Spraggon G, Schultz PG, Wang F (2009a) Genetic incorporation of a metal-ion chelating amino acid into proteins as a biophysical probe. J Am Chem Soc 131:2481–2483
Lee HS, Guo J, Lemke EA, Dimla RD, Schultz PG (2009b) Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae. J Am Chem Soc 131:12921–12923
Lee YJ, Wu B, Raymond JE, Zeng Y, Fang X, Wooley KL, Liu WR (2013) A genetically encoded acrylamide functionality. ACS Chem Biol 8:1664–1670
Lemke EA, Summerer D, Geierstanger BH, Brittain SM, Schultz PG (2007) Control of protein phosphorylation with a genetically encoded photocaged amino acid. Nat Chem Biol 3:769–772
Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444
Plass T, Milles S, Koehler C, Schultz C, Lemke EA (2011) Genetically encoded copper-free click chemistry. Angew Chem Int Ed 50:3878–3881
Schmidt MJ, Borbas J, Drescher M, Summerer D (2014) A genetically encoded spin label for electron paramagnetic resonance distance measurements. J Am Chem Soc 136:1238–1241
Seitchik JL, Peeler JC, Taylor MT, Blackman ML, Rhoads TW, Cooley RB, Refakis C, Fox JM, Mehl RA (2012) Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes. J Am Chem Soc 134:2898–2901
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234
Wang L, Schultz PG (2005) Expanding the genetic code. Angew Chem Int Ed 44:34–66
Wang J, Xie J, Schultz PG (2006) A genetically encoded fluorescent amino acid. J Am Chem Soc 128:8738–8739
Xie J, Liu W, Schultz PG (2007) A genetically encoded bidentate, metal-binding amino acid. Angew Chem Int Ed 46:9239–9242
Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S (2008) Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem Biol 24:1187–1197
Young TS, Ahmad I, Yin JA, Schultz PG (2010) An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol 395:361–374
Acknowledgments
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2014003870).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Handling Editor: S. Murch.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Ko, W., Kim, S., Jo, K. et al. Genetic incorporation of recycled unnatural amino acids. Amino Acids 48, 357–363 (2016). https://doi.org/10.1007/s00726-015-2087-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-015-2087-x