Effect of energy source on the efficiency of translational termination during cell-free protein synthesis.
We studied how the fidelity of translation termination is affected by the method of ATP regeneration during cell-free protein synthesis. During the in vivo expression of hEPO, whose termination is directed by the UGA codon, we found that substantial proportions of the translational products showed a larger molecular weight than expected. Similar results were obtained in a cell-free synthesis reaction using phosphoenol pyruvate (PEP) or 3-phosphoglycerate (3PG) for ATP regeneration. However, when the energy source was switched to creatine phosphate (CP), the readthrough of the UGA codon was completely repressed and only the target protein of the correct size was expressed in a high yield. To the best of our knowledge, this is the first report describing the relationship between the regeneration of nucleotide triphosphates and protein readthrough, and we also believe that the discovery would pave the way to the selective and efficient expression of target proteins in cell-free protein synthesis systems.
Ahn
Jin-Ho
JH
Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, Seoul 151-742, Republic of Korea.
Choi
Cha-Yong
CY
Kim
Dong-Myung
DM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D002474:Cell-Free System; D018388:Codon, Terminator; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics; D005988:Glyceric Acids / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010443:Peptide Chain Termination, Translational; D010725:Phosphocreatine / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism
D018388:Codon, Terminator; D005988:Glyceric Acids; D010725:Phosphocreatine; D004921:Erythropoietin; D010728:Phosphoenolpyruvate; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate