ExpressionandpurificationofRab8A(1-181)stoichiometrically phosphorylatedatpThr72 (theLRRK2site) v1

A subset of small GTPases of the Rab family including Rab8A (Uniprot: P61006) have been identified as substrates of the Leucine Rich Repeat Kinase 2 (LRRK2; Uniprot Q5S007)) and the Protein Phosphatase PPM1H (Uniprot Q9ULR3) (Steger et al., 2016; Berndsen et al., 2019). In order to perform detailed study of Rab phosphorylation and dephosphorylation, as well as produce phosphorylation site-specific antibodies, and to carry out drug discovery screens, it is necessary to produce hundreds of milligrams of pure, stoichiometrically phosphorylated Rab8A protein. The full-length sequence of Rab8A is prone to aggregation and precipitation when expressed in bacteria. Therefore, a shorter fragment, spanning residues 1-181 is more useful for large scale expression. Here we describe in detail the method we use to produce milligram quantities of stoichiometrically Thr72 phosphorylated Rab8A[1-181]. We employ the MST3 kinase to phosphorylate Rab8A at Thr72, as this kinase is much easier and less expensive to produce or purchase than LRRK2 (Berndsen et al., 2019, Vieweg et al. 2020).