Elsevier

Biotechnology Advances

Volume 32, Issue 3, May–June 2014, Pages 564-574
Biotechnology Advances

Research review paper
Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants

https://doi.org/10.1016/j.biotechadv.2014.02.003Get rights and content

Abstract

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.

Introduction

Functionally diverse group of membrane proteins anchored in cellular membrane, like G protein-coupled receptors (GPCRs), are critical for signal transduction in cell physiological actions (Ballesteros and Palczewski, 2001, Stefanovic and Hegde, 2007). The receptors receive primary stimuli such as hormone, odorant, light and neurotransmitter, producing vision, smell, taste and pain by an exogenous signal (Premont and Gainetdinov, 2007, Rosenbaum et al., 2009), associated with the extracellular and intracellular signal transmissions. GPCRs represent the most important family of drug targets, being responsible for more than 60% of the current pharmaceutical drugs for a variety of diseases (Gainetdinov et al., 2004, Lagerstrom and Schioth, 2008, McCusker et al., 2007). However, their structure/function is still difficult to investigate due to low abundance of GPCRs on cell surface. Heterogeneous protein synthesis systems, including Escherichia coli (E. coli) system, yeast system, mammalian or insect cells with virus vector expression system, cell-free system and zebrafish system (Ghaemmaghami et al., 2003, Sarramegna et al., 2003), contribute to high yields of GPCRs from micrograms to milligrams, but are still with the post-translation modification problems such as phosphorylation, glycosylation and N-myristoylation (Carlson et al., 2011, Gerngross, 2004, Prinster et al., 2005).

Compared with those various expression systems, cell-free system has some advantages to produce some notorious proteins (Katzen et al., 2005, Nirenberg and Matthaei, 1961, Spirin, 2004) or construct proteins at industrial scale, but still are limited to the low protein production rate, expensive reagent costs and short reaction durations of protein synthesis (Carlson et al., 2011, Zawada et al., 2011).

Stabilization of the GPCRs is often a vital issue. The traditional detergents SDS (Sodium dodecyl sulfate), DDM (n-dodecyl-β-d-maltoside) and Triton X-100 have been widely used in these studies. Self-assembly peptide surfactants as a new kind of detergent, coupled with cell-free systems, might also be with potential ability to stabilize membrane proteins (Heerklotz et al., 2009, Le Maire et al., 2008), like olfactory receptors (ORs), human trace amine-associated receptor (TAAR) and human vomeronasal receptors (VNRs) (Corin et al., 2011a, Corin et al., 2011b, Luo and Zhang, 2012). In this review, we will focus on the protein synthesis systems, the surfactants of stabilizing membrane proteins and some typical GPCRs using cell-free system with self-assembling peptide detergent to review their progress in recently years.

Section snippets

Expression

The available sufficient quantities of purified proteins are one important factor to obtain high-resolution 3D structures of GPCRs. Impractical rendering of their direct purification from the poorly abundance natural sources (Sarramegna et al., 2003), recombination heterologous expression systems with the development of protein synthesis technologies has circumvented the problem, including E. coli expression system, yeast expression system, mammalian or insect cells with virus vector expression

Cell-free and GPCRs

Protein synthesis for configuration study in cell-free expression system has been performed worldwide with cost-efficient and other advantages: 1) Reaction can be proceeded in a short time; 2) Conditions of reaction can be convenient to control with post-translational modifications; 3) Plasmid or DNA can be directly used for protein expression; 4) Special proteins can be expressed with a composition of non-natural amino acids (Carpenter et al., 2008, McCusker et al., 2007, Nirenberg and

Photosystem I and II

The peptide detergents directly interact with the hydrophobic domains of membrane proteins to increase their solubility, significantly preventing the degradation or precipitation of proteins. These peptides not only replace the intact proteins from phospholipid bilayer to increase solubilization, but also maintain the proteins' structural integrity with biological function. CD spectrum shows that the novel peptide detergents can essentially stabilize photosystem complex during purification (

Self-assembly peptide surfactants coupled with cell-free technology to GPCR

Much efforts have been undertaken to solve the bottlenecks in prokaryotic and eukaryotic expression systems, and some achievements have been acquired, especially the protein synthesis system of cell-free, which is crucial not only for expressing proteins without toxicity to host cells, but also for producing appreciated yields with potential post-translational modifications (Kai et al., 2012, Lyukmanova et al., 2012, Matthies et al., 2011).

Cell-free protein synthesis system is a robust method

Summary and perspective

Membrane proteins play vital roles in all living systems. They are involved in energy conversions, cell–cell and cell-environmental communications and sensing, specific ion channels and pump transports, and all sorts of transports. Membrane proteins are also essential for sight, hearing, smell, taste, touch and temperature sensing of human beings, and GPCRs are specially crucial in learning, memory, stem cell renewal and differentiation, body-plan development, immune system, aging and more.

Acknowledgments

ZL was supported in part by China National “985 Project” and by the grant from the National Natural Science Foundation of China (NSFC 81101417), the Natural Science Foundation Project of CQCSTC (2011BB5134), and the Specialized Research Fund for the Doctoral Program of Higher Education (20115503120010).

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