Expression of Reticulomyxa filosa alpha- and beta-tubulins in Escherichia coli yields soluble and partially correctly folded material.
Tubulins are highly conserved multidomain proteins that have to interact with eukaryotic chaperonins to gain their correct three-dimensional conformation. The prokaryotic chaperonin system of GroEL/ES is able to generate intermediate folding states but not natively folded tubulin. To create a system for studying these folding intermediates, tubulins from the giant amoeba Reticulomyxa filosa (alpha 2- and beta 2-tubulin) were expressed in Escherichia coli singly or in tandem. In all cases, soluble tubulin was generated in amounts of 5-10 mg/l culture. This is the first reported expression of soluble tubulin in bacterial cells. Of particular interest was the observation that upon coexpression with R. filosa beta 2-tubulin, proteolytic degradation of alpha 2-tubulin was reduced and more full-length product remained intact. This observation points to a specific interaction of alpha 2- and beta 2-tubulins in the E. coli cell. The sites of interaction are most probably the same that are responsible for the binding of native alpha 2- and beta 2-tubulin. The established expression system therefore seems well suited for further studies concerning the folding of tubulins.
Linder
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Adolf-Butenandt-Institute/Cell Biology, Munich, Germany. stefan.linder@klp.med.uni-muenchen.de
Schliwa
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M
Kube-Granderath
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E
D000656:Amoeba / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D018835:Chaperonin 10 / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D014404:Tubulin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
D018835:Chaperonin 10; D018834:Chaperonin 60; D017931:DNA Primers; D011994:Recombinant Proteins; D014404:Tubulin; D010450:Endopeptidases