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Site‐Specific Chemical Modification of Proteins with a Prelabelled Cysteine Tag Using the Artificially Split Mxe GyrA Intein
Author(s) -
Kurpiers Thomas,
Mootz Henning D.
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800319
Subject(s) - intein , cysteine , protein splicing , protein tag , nonribosomal peptide , biochemistry , chemistry , peptide , chemical biology , cyclic peptide , trans splicing , fusion protein , rna splicing , biosynthesis , gene , enzyme , recombinant dna , rna
The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine‐containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl‐reactive probe. In a second step, protein trans‐splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein‐labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site‐specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine‐reactive probes with a straightforward route for their site‐specific incorporation even into complex and cysteine‐rich proteins.