Abstract
The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is then purified in a buffer containing dodecyl maltoside using Strep-Tactin affinity chromatography. Following enzymatic cleavage of the attached GFP and Strep-tag by thrombin, the P2X7 receptor is isolated using size exclusion chromatography. This method typically yields ~2 mg of purified protein from 6 L of Sf9 culture. Purified protein can be stored in a buffer containing 15% glycerol at 4 °C for at least 2 months and used for a variety of functional and structural studies (Karasawa and Kawate, 2016).
Keywords: P2X7, Sf9, Baculovirus expression system, Eukaryotic membrane protein
Background
The P2X7 receptor is one of the seven subtypes of the purinergic P2X receptor family and has been a promising novel drug target for a wide range of diseases such as neurodegenerative disorders, epilepsy, and neuropathic pain (North and Jarvis, 2013; Bhattacharya and Biber, 2016). Despite the well-documented clinical relevance, mechanisms underlying P2X7 specific functions are unclear. For example, it remains controversial whether P2X7 itself converts into a large pore or if P2X7 activation leads to an opening of another large-conductance channel such as pannexin1 (Alves et al., 2014).To unambiguously unravel the P2X7 receptor specific mechanisms, it is desirable to investigate the properties of this membrane channel in vitro in the absence of other proteins. It is also extremely advantageous to capture snapshots of the P2X7 receptor conformations throughout its gating cycle using techniques like X-ray crystallography and cryo-electron microscopy. However, purification of eukaryotic membrane proteins is nontrivial due to low expression levels and instability in detergents. Furthermore, complex folding mechanisms and necessary post-translational modifications force researchers to use eukaryotic host cells, which is time and cost consuming. Though several laboratories have established their own protocols for purifying eukaryotic membrane proteins using insect cells (Karakas et al., 2011; Hattori and Gouaux, 2012), it is often challenging to mimic experimental conditions simply based on the methods reported in research papers, which normally lack tips and special notes due to space limitation. This protocol aims to provide an in-depth guide for expressing and purifying eukaryotic membrane proteins using an insect cell/baculovirus system. Based on this protocol, we have successfully purified milligram quantities of a mammalian P2X7 receptor, which we used to determine its crystal structures (Karasawa and Kawate, 2016).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Quality of the purified P2X7 receptor can be analyzed by SEC and SDS-PAGE (Figure 11). Figure 11A shows a representative SEC profile with a single P2X7 peak, suggesting that the purified P2X7 receptor is monodisperse. A representative SDS-PAGE gel image (Figure 11B) verifies the chemical purity of this sample. Figure 11. Characterization of purified P2X7. Representative SEC profile (A) and a gel image (B) of purified P2X7.
Notes
Recipes
Acknowledgments
We thank K. Michalski and P. Nguyen for critical comments. This protocol was adapted from our previous work (Karasawa and Kawate, 2016). This work was supported by the National Institutes of Health (GM114379 and NS072869).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.