Cell-free N-terminal protein labeling using initiator suppressor tRNA.
A highly efficient method for the introduction of fluorophores and other markers at the N terminus of proteins produced in a cell-free extract has been developed. The method utilizes an amber (CUA) initiator suppressor tRNA chemically aminoacylated with a fluorophore-amino acid conjugate which is introduced into an Escherichia coli S30 cell-free translation system. The DNA template contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon. Using this approach, the fluorophore BODIPY-F1 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-propionic acid) has been incorporated at the N terminus of several model proteins. The specific labeling achieved (27-67%) using this approach is much higher than that of wild-type tRNAs. Several potential biophysical and biotechnological applications of this new technology are described.
Mamaev
Sergey
S
AmberGen, Inc., 1106 Commonwealth Avenue, Boston, MA 02215, USA.
Olejnik
Jerzy
J
Olejnik
Edyta Krzymanska
EK
Rothschild
Kenneth J
KJ
D000215:Acylation; D000586:Amination; D001896:Boron Compounds; D002138:Calibration; D002474:Cell-Free System; D003062:Codon / Q000235:genetics; D004926:Escherichia coli; D011422:Propionates / Q000737:chemistry; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013698:Templates, Genetic
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D003062:Codon; D011422:Propionates; D011506:Proteins; D012358:RNA, Transfer, Met; C029658:propionic acid