Abstract
Improved ways to cleave peptide chains at engineered sites easily and
specifically would form useful tools for biochemical research. Uses of such
methods include the activation or inactivation of enzymes or the removal of
tags for enhancement of recombinant protein expression or tags used for
purification of recombinant proteins. In this work we show by gel
electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be
used to cleave fusion proteins specifically at sites where sequences of His
residues have been introduced by protein engineering. The His residues could
be either consecutive or spaced with other amino acids in between. The
cleavage reaction required the presence of low concentrations of ascorbate and
in the case of Cu(II) also hydrogen peroxide. The amount of metal ions
required for cleavage was very low; in the case of Cu(II) only one to two
molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10
molar equivalents gave optimal cleavage. The reaction occurred within
minutes, at a wide pH range, and efficiently at temperatures ranging from 0°C
to 70°C. The work described here can also have implications for understanding
protein stability in vitro and in vivo.
Original language | English |
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Pages (from-to) | 1751-1761 |
Journal | Protein Science |
Volume | 16 |
Issue number | 8 |
DOIs | |
Publication status | Published - 2007 |
MoE publication type | A1 Journal article-refereed |
Keywords
- protein cleavage
- His tag
- artificial protease
- protein stability
- Co(II)
- Cu(II)