TY - JOUR
T1 - Comparative evaluation of two cell-free protein synthesis systems derived from Escherichia coli for genetic code reprogramming
AU - Lee, Ki Baek
AU - Kim, Ho Cheol
AU - Kim, Dong Myung
AU - Kang, Taek Jin
AU - Suga, Hiroaki
PY - 2012/12/5
Y1 - 2012/12/5
N2 - Genetic codes can be reprogrammed to code for non-proteinogenic amino acids during protein synthesis. Technologically, these non-proteinogenic amino acids are incorporated into proteins by artificially charging them to suppressor-tRNAs that can reprogram the existing codons. Several methods and systems for genetic code reprogramming have been reported including methods for charging non-proteinogenic amino acids to tRNA molecules, codons for reprogramming, and systems for protein synthesis. However, there has been no systematic, comparative evaluation of cell-free protein synthesis systems in genetic code reprogramming for their efficiencies and robustness even with their potential usefulness in the field. Here we compare two cell-free protein synthesis systems, the crude S12 and PURE system, with the codon systems, non-proteinogenic amino acids, and the positions in the protein for reprogramming as variables. We show that the combined use of CCCG four-nucleotide codon that is newly developed in this study and the crude S12 system is the most reliable and robust method of choice, while the use of traditional UAG amber stop codon along with an RNA aptamer toward peptide release factor 1 can yield the most plentiful product with certain variations.
AB - Genetic codes can be reprogrammed to code for non-proteinogenic amino acids during protein synthesis. Technologically, these non-proteinogenic amino acids are incorporated into proteins by artificially charging them to suppressor-tRNAs that can reprogram the existing codons. Several methods and systems for genetic code reprogramming have been reported including methods for charging non-proteinogenic amino acids to tRNA molecules, codons for reprogramming, and systems for protein synthesis. However, there has been no systematic, comparative evaluation of cell-free protein synthesis systems in genetic code reprogramming for their efficiencies and robustness even with their potential usefulness in the field. Here we compare two cell-free protein synthesis systems, the crude S12 and PURE system, with the codon systems, non-proteinogenic amino acids, and the positions in the protein for reprogramming as variables. We show that the combined use of CCCG four-nucleotide codon that is newly developed in this study and the crude S12 system is the most reliable and robust method of choice, while the use of traditional UAG amber stop codon along with an RNA aptamer toward peptide release factor 1 can yield the most plentiful product with certain variations.
KW - Acetyllysine
KW - Cell-free protein synthesis
KW - Flexizyme
KW - Genetic code reprogramming
KW - Methyllysine
KW - Non-proteinogenic amino acid
UR - http://www.scopus.com/inward/record.url?scp=84875265471&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2013.01.011
DO - 10.1016/j.jbiotec.2013.01.011
M3 - Article
C2 - 23395618
AN - SCOPUS:84875265471
SN - 0168-1656
VL - 164
SP - 330
EP - 335
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 2
ER -