Overexpression, purification, and functional analysis of recombinant human tubulin dimer

Abstract

a b s t r a c tMicrotubules consisting of tubulin dimers play essential roles in various cellular functions. Investigating the structure-function relationship of tubulin dimers requires a method to prepare sufficient quantities of recombinant tubulin. To this end, we simultaneously expressed human a1-and b3-tubulin using a baculovirus-insect cell expression system that enabled the purification of 5 mg recombinant tubulin per litre of cell culture. The purified recombinant human tubulin could be polymerized into microtubules that glide on a kinesin-coated glass surface. The method provides a powerful tool for in vitro functional analyses of microtubules.

The tubulin code at a glance

“…recombinant tubulin with controlled PTMs -in order to study the mechanisms of the tubulin code in vitro. The first exciting advances in this direction have recently been made (Barisic et al, 2015;Minoura et al, 2013;Pamula et al, 2016;Sirajuddin et al, 2014;Vemu et al, 2016).…”

Writing and Reading the Tubulin Code

“…Although microtubules in cells show topographically defined modification patterns, the isolation procedure of microtubules from brain tissue results in complete scrambling of all the tubulin modifications and isoforms and thus makes the task of deciphering a tubulin code impossible. Recent advances now allow the purification of naive, unmodified tubulin from various sources (9, 10) as well as recombinant tubulin (11,12) in which posttranslational modification sites can be mutated. Using unmodified human tubulin, we have shown how to generate defined posttranslationally modified tubulin and microtubules that are tyrosinated, glutamylated, and acetylated (13).…”

“…Although the field has made tremendous progress in recent decades identifying a compendium of microtubule-interacting proteins and understanding their interplay and regulation in the cell, we are just now starting to unravel the basic mechanisms used by cells to chemically modify microtubules, despite the fact that tubulin posttranslational modifications have been known for over 40 years. A renaissance of interest into the roles of tubulin posttranslational modifications has been precipitated by the discovery in the last few years of the enzymes responsible for these modifications (3)(4)(5)(6)(7)(8), methods for producing unmodified (9,10), engineered, (11,12) as well as chemically defined modified tubulin (13), and developments and refinements of in vitro microtubule-based assays using high-resolution microscopy and microfabricated substrates (14 -16).…”

Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel

“…The results of these studies (3,(32)(33)(34)(35) demonstrate that recombinant tubulins are reliable means to study tubulin isotype-specific effects in vitro and to dissect the role of individual tubulin's CTTs.…”

“…fully functional and able to polymerize and form microtubules suitable for motor protein motility and processivity (3,(33)(34)(35). The recombinant human homogeneous microtubules showed different rates of polymerization compared with wild type (32,35).…”