Properties of microtubules assembled from mammalian tubulin synthesized inEscherichia coli

When isolated from tissues, the alpha beta -dimeric protein tubulin consist s of multiple isoforms which originate from the expression and subsequent p osttranslational modification of multiple polypeptide sequences. Microtubul es studied in vitro consist of mixtures of these isoforms. It is therefore not known whether dimers composed of single sequences of alpha- and beta -t ubulin can polymerize to form microtubules, or whether posttranslational mo difications may be necessary for microtubule assembly. To initiate investig ation of these questions, rabbit reticulocyte lysate, which contains the cy toplasmic chaperonin CCT and its cofactors, was employed to prepare substan tial quantities (tens of micrograms) of active tubulin by in vitro folding of mouse alpha- and beta -tubulins recombinantly synthesized in E. coli. Th is recombinant tubulin is composed of only a single alpha -chain and a sing le beta -chain. When analyzed after folding by isoelectric focusing, each c hain yielded only one band, indicating that neither was detectably posttran slationally modified in the course of the folding reaction. When subjected to assembly-promoting conditions, this tubulin formed microtubules without the addition of any exogenous protein, Electron microscopy showed them to b e of normal morphology. Analysis of their protein composition showed that t hey are composed nearly entirely of recombinant tubulin. These results demo nstrate that the naturally occurring mixtures of isoforms are not strictly required for the formation of microtubules, They also open a route to other studies, both biomedical and structural, of fully defined tubulin in vitro .