Effect of temperature and ATP supply on the efficiency of programmed nonsense suppression.
Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce S-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.
Ahn
Jin-Ho
JH
Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, Seoul 151-742, Korea.
Kim
Nam-Young
NY
Kim
Tae-Wan
TW
Son
Jeong-Mi
JM
Kang
Taek-Jin
TJ
Park
Chang-Gil
CG
Choi
Cha-Yong
CY
Kim
Dong-Myung
DM
D000926:Anticodon / Q000737:chemistry*; D001483:Base Sequence; D002474:Cell-Free System / Q000737:chemistry; D018389:Codon, Nonsense / Q000737:chemistry*; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry*; D004921:Erythropoietin / Q000096:biosynthesis*; D004926:Escherichia coli / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis
D000926:Anticodon; D018389:Codon, Nonsense; D004921:Erythropoietin; D003545:Cysteine