title: A simple in vivo assay for increased protein solubility.
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.
authors.affiliation: Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
authors.forename: K L
authors.initials: KL
authors.lastname: Maxwell
authors.forename: A K
authors.initials: AK
authors.lastname: Mittermaier
authors.forename: J D
authors.initials: JD
authors.lastname: Forman-Kay
authors.forename: A R
authors.initials: AR
authors.lastname: Davidson
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002701:Chloramphenicol / Q000737:chemistry; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D019427:HIV Integrase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012995:Solubility
chemical_list: D011993:Recombinant Fusion Proteins; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D019427:HIV Integrase