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A simple in vivo assay for increased protein solubility

Published online by Cambridge University Press:  01 September 1999

KAREN L. MAXWELL
Affiliation:
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
ANTHONY K. MITTERMAIER
Affiliation:
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada Structural Biology and Biochemistry Program, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
JULIE D. FORMAN-KAY
Affiliation:
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada Structural Biology and Biochemistry Program, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
ALAN R. DAVIDSON
Affiliation:
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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Abstract

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.

Type
FOR THE RECORD
Copyright
© 1999 The Protein Society

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