Site-specific protein labeling by intein-mediated protein ligation.
Intein-mediated protein ligation (IPL) employs an intein to create a protein possessing a C-terminal thioester that can be ligated to a protein or peptide with an amino-terminal cysteine via a native peptide bond. Here we present a procedure to conduct isolation and labeling of recombinant proteins expressed in E. coli using synthetic short peptides possessing a fluorescent moiety. This approach can be readily utilized for site-specific conjugation of a fluorophore to the C-terminus of a protein of interest, without the drawback of non-specific chemical labeling. This chapter also gives a general review of the critical parameters of intein-mediated cleavage and ligation reactions.
New England BioLabs, Ipswich, MA 01938, USA. ghosh@neb.com
Inca
I
Ghosh
Nancy
N
Considine
Elissa
E
Maunus
Luo
L
Sun
Aihua
A
Zhang
John
J
Buswell
Thomas C
TC
Evans
Ming-Qun
MQ
Xu
D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D047668:Inteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013440:Sulfides / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D013440:Sulfides