Low solubility is a major stumbling block in the
detailed structural and functional characterization of
many proteins and isolated protein domains. The production
of some proteins in a soluble form may only be possible
through alteration of their sequences by mutagenesis. The
feasibility of this approach has been demonstrated in a
number of cases where amino acid substitutions were shown
to increase protein solubility without altering structure
or function. However, identifying residues to mutagenize
to increase solubility is difficult, especially in the
absence of structural knowledge. For this reason, we have
developed a method by which soluble mutants of an insoluble
protein can be easily distinguished in vivo in Escherichia
coli. This method is based on our observation that
cells expressing fusions of an insoluble protein to chloramphenicol
acetyltransferase (CAT) exhibit decreased resistance to
chloramphenicol compared to fusions with soluble proteins.
We found that a soluble mutant of an insoluble protein
fused to CAT could be selected by plating on high levels
of chloramphenicol.