Rab GTPases are essential mediators of membrane traffic, playing a role in the majority of cellular traffic of vesicles and organelles. Rab GTPases are conventionally considered active when GTP-bound and inactive in their GDP-bound form. In its GDP bound form, newly synthesised Rab GTPases undergo geranylgeranylation which directs membrane attachment. Rab GTPase activating proteins (GAPs) catalyze hydrolysis of the GTP bound to active Rab GTPases to inactivate them. However, currently there are no RabGAP-Rab GTPase pairs characterised in plants.
Previous work showed that Rab3GAP negatively regulates autophagy and increases host susceptibility to the oomycete pathogen Phytophthora infestans. In vivo studies confirmed that Rab8a and Rab3GAP interact with each other and Rab8a contributes positively to resistance against P. infestans. The primary objective of this project was to prepare a protein purification protocol for the potato Rab3Gap and Rab8a proteins for future in vitro GAP activity assays to test whether Rab3GAP functions as a GAP for Rab8a.
I cloned truncated forms of Rab8a, excluding residues responsible for its geranylgeranylation motif (CCXX) and membrane localisation domain at the c terminal. This truncated version of Rab8a was hypothesised to be easier to purify as it is expected to be more soluble, and thus present in higher amount in the soluble fraction instead of insoluble fraction (cell membranes or aggregates).
I cloned the wild-type Rab3GAP as well as Rab3GAP’s catalytic mutant and GAP domain only (Rab3GAP, Rab3GAPCM, GAP, GAPCM) alongside truncated wild-type Rab8a and truncated Rab8a-GDP mutant into vectors from pOPIN vector suite (pOPINS3C) using Gibson assembly. Small-scale overexpression tests were conducted for each protein prior to large-scale protein overexpression and protein purification using fast protein liquid chromatography (FPLC) to test whether the induction conditions were optimal for the expression of our protein of interest. Protein samples obtained from large-scale protein purifications were analysed by SDS-PAGE and stained with Coomassie-Blue.
I further cloned Rab3GAP, Rab3GAPCM, GAP and GAPCM in another vector from pOPIN vector suite (pOPINM) while truncated Rab8a and Rab8a-GDP in pOPINA. These two vectors carry different antibiotic resistant genes. When both types of vectors are used to co-transformed E. coli, media containing the two different antibiotics will force E. coli to retain both vectors and allow co-expression after induction. These vectors contain specific fusion proteins that aid protein production and solubility. pOPINS3C vector contains a small ubiquitine-like modifier protein (SUMO) tag that enhances protein solubility. Similarly, pOPINM carries a maltose binding protein (MBP) tag at the N-terminal of fusion protein that had been shown to be capable of increasing protein solubility and increasing protein production.
This project provided me with the opportunity to gain practical experience and knowledge in protein purification and molecular cloning techniques. I am grateful for the support from Dr Tolga Bozkurt for the BSPP bursary application and his guidance during this placement. I am also thankful for the day-to-day guidance from my lab mentor, Alexandre Leary as well as fellow lab members throughout this project.
Yow Rui Jin
Imperial College London