A simple in vivo assay for increased protein solubility

Low solubility is a major stumbling block in the detailed structural and fu nctional characterization of many proteins and isolated protein domains. Th e production of some proteins in a soluble form may only be possible throug h alteration of their sequences by mutagenesis. The feasibility of this app roach has been demonstrated in a number of cases where amino acid substitut ions were shown to increase protein solubility without altering structure o r function. However, identifying residues to mutagenize to increase solubil ity is difficult, especially in the absence of structural knowledge. For th is reason, we have developed a method by which soluble mutants of an insolu ble protein can be easily distinguished in vivo in Escherichia coli. This m ethod is based on our observation that cells expressing fusions of an insol uble protein to chloramphenicol acetyltransferase (CAT) exhibit decreased r esistance to chloramphenicol compared to fusions with soluble proteins. We found that a soluble mutant of an insoluble protein fused to CAT could be s elected by plating on high levels of chloramphenicol.