The thiol protease, papain (Mr = 23 kD according to Boehringer)
has a single unmodified cysteine in its binding pocket. The
enzyme may need to be pre-treated with a reducing agent to
make sure the thiol is available. Papain is also available from
Sigma and other biologic reagent companies.
Although not as readily available, the antibody fragment Fab' prepared from rabbit IgG comes to mind as a ~25kD protein
with a single cysteine in the 'hinge region'. There is a body of
literature on the modification of Fab' hinge region cysteine thiols
for use as immunotargeting therapeutic and diagnostic agents.
The preparation of Fab' would involve mild reduction of the
F(ab')2 fragment of IgG and removal of the reducing agent.
Mild reduction under neutral buffer conditions is necessary
so as to avoid reduction of intra-chain disulfide bonds.
Fab' derived from other species have varying numbers of
hinge region cysteines (e.g. 3 for mouse IgG1, although it is
possible to end up with a single free thiol there).
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Margaret E. (Peggy) Schott
Synthesis & Sequencing Shared Resource
E401 Research Bldg, Georgetown University
3970 Reservoir Rd, NW, Washington, DC
(202) 687-1684 FAX (202) 687-7505
Email: Schottm@gunet.georgetown.edu
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