Site-specific biosynthetic incorporation of a fluorescent tag into proteins via cysteine-tRNA(Cys).
Site-specific incorporation of biophysical probes into proteins during translation can permit structure/function studies on selected proteins in heterogeneous environments. We report here a procedure for incorporating a fluorescent tag into proteins via Escherichia coli Cys-tRNA(Cys) during in vitro protein synthesis. Naturally occurring Cys-tRNA(Cys) is an attractive vehicle for fluorophore incorporation since it can be readily prepared in quantity and reacted with commercially available fluorophores. Moreover, proteins can often be constructed with a single Cys so that fluorophore incorporation results in a tag at a unique site.
Department of Chemistry, University of Toronto, Ont., Canada.
Linda
L
Lien
P
P
Ananda
K
K
Seneviratne
Anna S I
AS
Jaikaran
G
G
Andrew Woolley
D001483:Base Sequence; D001896:Boron Compounds / Q000378:metabolism*; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012350:RNA, Transfer, Cys / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D011506:Proteins; D012350:RNA, Transfer, Cys