TY - JOUR
T1 - Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli.
AU - Galindo Casas, Meritxell
AU - Stargardt, Patrick
AU - Mairhofer, Juergen
AU - Wiltschi, Birgit
PY - 2020/11/20
Y1 - 2020/11/20
N2 - The site-specific incorporation of noncanonical amino acids (ncAAs) into proteins by amber stop codon suppression has become a routine method in academic laboratories. This approach requires an amber suppressor tRNACUA to read the amber codon and an aminoacyl-tRNA synthetase to charge the tRNACUA with the ncAA. However, a major drawback is the low yield of the mutant protein in comparison to the wild type. This effect primarily results from the competition of release factor 1 with the charged suppressor tRNACUA for the amber codon at the A-site of the ribosome. A number of laboratories have attempted to improve the incorporation efficiency of ncAAs with moderate results. We aimed at increasing the efficiency to produce high yields of ncAA-functionalized proteins in a scalable setting for industrial application. To do this, we inserted an ncAA into the enhanced green fluorescent protein and an antibody mimetic molecule using an industrial E. coli strain, which produces recombinant proteins independent of cell growth. The controlled decoupling of recombinant protein production from cell growth considerably increased the incorporation of the ncAA, producing substantially higher protein yields versus the reference E. coli strain BL21(DE3). The target proteins were expressed at high levels, and the ncAA was efficiently incorporated with excellent fidelity while the protein function was preserved.
AB - The site-specific incorporation of noncanonical amino acids (ncAAs) into proteins by amber stop codon suppression has become a routine method in academic laboratories. This approach requires an amber suppressor tRNACUA to read the amber codon and an aminoacyl-tRNA synthetase to charge the tRNACUA with the ncAA. However, a major drawback is the low yield of the mutant protein in comparison to the wild type. This effect primarily results from the competition of release factor 1 with the charged suppressor tRNACUA for the amber codon at the A-site of the ribosome. A number of laboratories have attempted to improve the incorporation efficiency of ncAAs with moderate results. We aimed at increasing the efficiency to produce high yields of ncAA-functionalized proteins in a scalable setting for industrial application. To do this, we inserted an ncAA into the enhanced green fluorescent protein and an antibody mimetic molecule using an industrial E. coli strain, which produces recombinant proteins independent of cell growth. The controlled decoupling of recombinant protein production from cell growth considerably increased the incorporation of the ncAA, producing substantially higher protein yields versus the reference E. coli strain BL21(DE3). The target proteins were expressed at high levels, and the ncAA was efficiently incorporated with excellent fidelity while the protein function was preserved.
KW - affibody
KW - amber suppression
KW - BL21(DE3) gp2
KW - genetic code expansion
KW - growth-decoupled protein production
KW - noncanonical amino acid
KW - Genetic code expansion
KW - Amber suppression
KW - Affibody
KW - Growth-decoupled protein production
KW - Noncanonical amino acid
UR - http://europepmc.org/abstract/med/33150786
UR - http://www.scopus.com/inward/record.url?scp=85096508932&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.0c00298
DO - 10.1021/acssynbio.0c00298
M3 - Article
C2 - 33150786
SN - 2161-5063
VL - 9
SP - 3052
EP - 3066
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 11
ER -