Fluorescent labeling of cell-free synthesized proteins by incorporation of fluorophore-conjugated nonnatural amino acids.
Although fluorescent dyes, such as fluorescein derivatives, have bulky and complex structures, nonnatural amino acids carrying these fluorescein derivatives are acceptable by the Escherichia coli ribosome and are useful for the cotranslational fluorescent labeling of cell-free synthesized proteins. Surprisingly, the incorporation efficiency of nonnatural amino acids carrying fluorescein derivatives into translated proteins depends on the source of the translational machinery used in cell-free protein synthesis. That is, whereas the E. coli ribosome efficiently supported the incorporation of nonnatural amino acids carrying fluorescein derivatives into a protein structure, no detectable fluorescent signal was observed from the protein expressed in the eukaryotic cell-free protein synthesis system performed in the presence of fluorescein-conjugated aminoacylated transfer RNA (tRNA).
Kang
Sang-Hyeon
SH
iNtRON Biotechnology, DCC-BIONET, Gyungki-do 462-120, Republic of Korea. kangsh0403@naver.com
Jun
Soo-Youn
SY
Kim
Dong-Myung
DM
D000215:Acylation; D000596:Amino Acids / Q000737:chemistry*; D002474:Cell-Free System; D005456:Fluorescent Dyes / Q000737:chemistry*; D008239:Lysine / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism
D000596:Amino Acids; D005456:Fluorescent Dyes; D012343:RNA, Transfer; D008715:Methionine; D008239:Lysine