Chemistry - A European Journal
10.1002/chem.201800910
FULL PAPER
d6) δ 173.6, 166.0, 164.3, 151.4, 150.0, 142.9, 141.8, 139.1, 132.4, 130.3,
Acknowledgements
1
21.6, 120.5, 111.8, 103.7, 48.8, 27.4, 20.8, 12.3. HRMS [M]+ calcd. for
37 41
[C H N
10]+: 625.3516, found: 625.3516.
Present work was supported by the Hungarian Scientific
Research Fund (OTKA, NN-116265) and the “Lendület” Program
of the Hungarian Academy of Sciences (LP2013-55/2013). EAL
acknowledges funding from the SPP1623. The authors thank the
Proteomics core facility (EMBL Heidelberg) for technical support.
Protein expression and labeling
Tagging experiment
We prepared
a Maltose binding protein domain (MBP) construct,
containing a FLAG-Tag as well as a short linker (KAEAADAEAAK) in front
of the MBP sequence. The FLAG-tag and the linker were cloned by
conventional overlap PCR and ligated into a pBAD-MBP-8His plasmid
using restriction enzymes. In addition we also cloned variants, which
harbored one or two Amber STOP codons instead of the lysine residues
in the short linker sequence (KAEAADAEAA-Amber) and Amber-
AEAADAEAA-Amber) by site directed mutagenesis. The short linker was
Keywords: bioorthogonal • fluorogenic probes • non-canonical
amino acid • two-point labeling • fluorogenic crosslinker
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(Invitrogen). Expression was done in 500 ml TB medium adding 100 µg/ml
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For the labeling reaction 50 pmol of GFPY39BCN were mixed with 50 pmol
of dye in a 10 µl reaction using 1×PBS to fill up the reaction mix. For each
time point, 1μl of the labeling reaction was mixed with 8 µl 1×PBS and 1 µl
[
10 mM BCN-Lysine, to stop the labeling reaction immediately. Each
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