文章基本信息

标题：Internal (His)6-tagging delivers a fully functional hetero-oligomeric class II chaperonin in high yield
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作者：Danielle M. Paul ; Fabienne Beuron ; Richard B. Sessions 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2016
卷号：6
期号：1
DOI：10.1038/srep20696
语种：English
出版社：Springer Nature
摘要：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.