the one in Coomassie blue staining (Fig. 5B). To test whether
the synthesized diubiquitin can be folded to its native
form, circular dichroism (CD) was measured with dialyzed
diubiquitin 8. The CD spectrum indicated that the diubiquitin
was well folded after dialysis (Fig. 5D). Next we performed the
cleavage assay using ubiquitin C-terminal hydrolase. It was
found that the diubiquitin can be hydrolyzed by the hydrolase,
UCH-L3 (Fig. S11, Electronic Supplementary Information).
In summary, herein we have optimized the dual native
chemical ligation approach by replacing the strong acid-labile
Cbz with the photolabile NVOC protecting group for the
side-chain amine of 4-mercaptolysine. The synthesis of
K48-linked diubiquitin using the improved protocol demon-
strates the practical utility of this ligation strategy in synthetic
protein chemistry.
Fig.
4 C4 analytic HPLC monitored ligation between 7 and
Ub(1–76)-MES at 0 h, 6 h and 10 h, respectively. Peak a: mixture of
7 and ubi(1–76)-MES. Peak b: ligation product. Peak c: mixture of
ubi(1–76)-OH and small amount of remaining 7 and ubi(1–76)-SBn.
Peak *: nonproteinous product. Note: 0 h HPLC was run before
adding benzyl mercaptan. Bn = benzyl.
This work was supported by A*Star of Singapore (BMRC
08/1/22/19/588 to C.-F.L.) and Nanyang Technological
University.
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native K48-linked diubiquitin. For the characterization
of the final product, the diubiquitin was checked with 18%
SDS-PAGE. Coomassie blue staining showed a single band.
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c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7199–7201 7201