title: In vivo increase of solubility of overexpressed Hha protein by tandem expression with interacting protein H-NS.
abstract: Gene cloning in appropriate vectors followed by protein overexpression in Escherichia coli is the common means for protein purification. This approach has many advantages but also some drawbacks; one of these is that many proteins fail to achieve a soluble conformation when overexpressed in E. coli. Hha protein belongs to a family of nucleoid-associated proteins functionally related to the H-NS family of proteins. Hha-like proteins and H-NS-like proteins are able to semidirectly bind to each other. We show in this work that overexpressed Hha or HisHha protein (a functional derivative of Hha containing a 6x His tag at the amino end) from a T7-polymerase promoter in BL21 DE3 E. coli strains results in the vast majority of the protein accumulated in insoluble aggregates (inclusion bodies). We also show that tandem overexpression of HisHha and H-NS increases the solubility of HisHha and prevents the formation of inclusion bodies. Single amino acid substitutions in the HisHha protein, which impair interaction with H-NS, render insoluble protein even when tandem-expressed with H-NS, tandem expression of an insoluble protein and an interacting partner is an experimental strategy which could be useful to increase the solubility of other overexpressed proteins in E. coli.
authors.affiliation: Department of Microbiology, Faculty of Biology, University of Barcelona, Avda. Diagonal, 645, 08028 Barcelona, Spain.
authors.forename: José I
authors.initials: JI
authors.lastname: Pons
authors.forename: Sonia
authors.initials: S
authors.lastname: Rodríguez
authors.forename: Cristina
authors.initials: C
authors.lastname: Madrid
authors.forename: Antonio
authors.initials: A
authors.lastname: Juárez
authors.forename: José M
authors.initials: JM
authors.lastname: Nieto
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C031908:H-NS protein, bacteria; C079209:hha protein, E coli