title: A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.
abstract: We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification.
authors.affiliation: Tropogen Inc., Privolnaya, 2, bld.1, G15, Moscow 109145, Russia.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Sharapova
authors.affiliation: Tropogen Inc., Privolnaya, 2, bld.1, G15, Moscow 109145, Russia.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Yurkova
authors.affiliation: Tropogen Inc., Privolnaya, 2, bld.1, G15, Moscow 109145, Russia anfedorov@yahoo.com.
authors.forename: Alexey N
authors.initials: AN
authors.lastname: Fedorov
mesh_terms: D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D012995:Solubility; D016963:Thermus thermophilus / Q000737:chemistry / Q000235:genetics*
chemical_list: D018834:Chaperonin 60; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine
keywords: insoluble proteins; minichaperone; protein fusion; protein stabilization; recombinant proteins