Specific Labeling of polypeptides at amino-terminal cysteine residues using Cy5-benzyl thioester

被引:34
作者
Schuler, B
Pannell, LK
机构
[1] NIDDKD, Chem Phys Lab, NIH, Bethesda, MD 20892 USA
[2] NIDDKD, Struct Mass Spectrometry Facil, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA
关键词
D O I
10.1021/bc025509t
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Even for moderately sized proteins, the multiple occurrence of cysteine and lysine residues often prevents the specific labeling of polypeptides with a single probe. To increase specificity, a method was developed to convert the commonly available succinimidyl esters of fluorescent dyes into benzyl thioesters via trimethyl aluminum-activated benzyl mercaptan. The thioester can then be reacted very specifically with polypeptides containing an N-terminal cysteine residue, forming a stable amide bond, analogous to the native chemical ligation of peptide fragments. Both reaction steps are easy to perform and proceed to high yields. The practicability of the approach was demonstrated using the popular cyanine dye Cy5 and a soluble peptide, and it is expected to be applicable to a wide range of succinimidyl esters and both chemically and recombinantly synthesized proteins. The method should dramatically facilitate the preparation of proteins for experiments requiring exact positioning of labels, for instance, Forster resonance energy transfer studies.
引用
收藏
页码:1039 / 1043
页数:5
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