title: Internal (His)₆-tagging delivers a fully functional hetero-oligomeric class II chaperonin in high yield.
abstract: Group II chaperonins are ATP-ases indispensable for the folding of many proteins that play a crucial role in Archaea and Eukarya. They display a conserved two-ringed assembly enclosing an internal chamber where newly translated or misfolded polypeptides can fold to their native structure. They are mainly hexadecamers, with each eight-membered ring composed of one or two (in Archaea) or eight (in Eukarya) different subunits. A major recurring problem within group II chaperonin research, especially with the hetero-oligomeric forms, is to establish an efficient recombinant system for the expression of large amounts of wild-type as well as mutated variants. Herein we show how we can produce, in E. coli cells, unprecedented amounts of correctly assembled and active αβ-thermosome, the class II chaperonin from Thermoplasma acidophilum, by introducing a (His)6-tag within a loop in the α subunit of the complex. The specific location was identified via a rational approach and proved not to disturb the structure of the chaperonin, as demonstrated by size-exclusion chromatography, native gel electrophoresis and electron microscopy. Likewise, the tagged protein showed an ATP-ase activity and an ability to refold substrates identical to the wild type. This tagging strategy might be employed for the overexpression of other recombinant chaperonins.
authors: School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol BS8 1TD, UK.
authors: Danielle M
authors: DM
authors: Paul
authors: Division of Structural Biology, The Institute of Cancer Research, London SW7 3RP, UK.
authors: Fabienne
authors: F
authors: Beuron
authors: School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors: Richard B
authors: RB
authors: Sessions
authors: School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.|Istituto di Chimica del Riconoscimento Molecolare, CNR c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, 00168 Roma, Italy.
authors: Andrea
authors: A
authors: Brancaccio
authors: School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors: Maria Giulia
authors: MG
authors: Bigotti
mesh_terms: D019843:Archaeal Proteins / Q000096:biosynthesis / Q000235:genetics; D056385:Group II Chaperonins / Q000096:biosynthesis / Q000235:genetics; D006639:Histidine / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013822:Thermoplasma / Q000235:genetics*
chemical_list: D019843:Archaeal Proteins; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D056385:Group II Chaperonins