A cell-free protein synthesis system as an investigational tool for the translation stop processes.
Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.
Kang
Taek Jin
TJ
School of Chemical Engineering, College of Engineering, Seoul National University, Seoul, South Korea.
Woo
Ji Hyoung
JH
Song
Hui Kyoung
HK
Ahn
Jin Ho
JH
Kum
Jae Wook
JW
Han
Jin
J
Choi
Cha Yong
CY
Joo
Hyun
H
D002474:Cell-Free System / Q000378:metabolism; D018388:Codon, Terminator / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*
D018388:Codon, Terminator; D012346:RNA, Transfer, Amino Acyl; D004921:Erythropoietin; D003545:Cysteine