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.
Highlights
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U group II chaperonins does range from that of the homo-oligomers and α β hetero-oligomers found in Archaebacteria to that of the hetero-oligomers of 8 different subunits found in Eukarya, where the complex is defined as CCT/TriC
In previous work we presented the first expression system for the production in E. coli of homogeneous, fully functional α β -hecadecamers of the thermosome from Thermoplasma acidophilum[11], an archaeal chaperonin composed of two rings of alternating α and β subunits whose structure has been solved at high resolution[5]
Summary
This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. We show how we can produce, in E. coli cells, unprecedented amounts of correctly assembled and active αβ-thermosome, e the class II chaperonin from Thermoplasma acidophilum, by introducing a (His)6-tag within a loop in the α subunit of the complex. In S. cerevisiae, an internal tag was introduced (within a loop) in the apical domain of CCT3/γ , which allowed the recovery of the whole complex through affinity purification[8], whereas the CCT4 and CCT5 human subunits have been tagged and expressed recombinantly in E. coli, and shown to be able to form active hexadecamers, in a homo-oligomeric form[9]. In order to measure the effect of the tagged thermosome on protein folding, we first identified a series of enzymes from Thermoplasma acidophilum whose activity could be recorded spectrophotometrically to use as possible substrates in refolding experiments and, where not available otherwise, we cloned and expressed them in E.coli (see Materials and Methods for details). The lag phase of the recovery in activity (not reported in the graph for clarity) is typically between 1 and 3 minutes and was not significantly affected by the presence of thermosome, either with or without ATP
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