Expression, Purification, and Microscopy-Based Assays for Engineered Recombinant Tyrosinated, Detyrosinated, and Δ2...

Microtubules are noncovalent polymers assembled from α/β tubulin dimers. Their structure, dynamics and interaction with effectors are regulated through the expression of diverse tubulin isotypes and chemically diverse posttranslational modifications, also known as the "tubulin code." Understanding the biophysical correlates between tubulin sequence, posttranslational modifications, and microtubule structure and dynamics requires the ability to engineer tubulin and produce homogenous, chemically well-defined tubulin preparations. Here, we provide a protocol for the baculovirus expression and three-step purification of recombinant α1A/βIII tubulin in its tyrosinated, detyrosinated, Δ2 form as well as α-tailless form. Our protocol yields milligrams of pure, homogenous, and monodisperse recombinant human tubulin suitable for structural studies and in vitro reconstitution assays. Our system allows facile engineering of diverse tubulin variants, providing a necessary tool for understanding microtubule structure and dynamics, and the effects of the tubulin code on microtubule functions.