Membrane proteins (MPs) represent one third of the total proteins encoded by the human genome. These include receptors, ion channels
, transporters, and porins. They play an important role in a wide range of biological processes like cell-to-cell communication, extracellular and intracellular ligand recognition, signal transduction, ion-channel conductance, and transport of a range of substrates across the membranes which are vital for survival of any organism. Any functional defect in the MPs could affect the cellular activities which could often lead to a wide range of diseases like Alzheimer’s disease, cystic fibrosis, epilepsy, cardia arrhythmia, and migraine [1,2,3]. Due to their medical importance, MPs have become more than 50% of the total drug targets from pharmaceutical companies. However, despite their significance in cellular physiology, complete structural information is only known for a small percentage of MPs. This is due to the lack of methods to synthesize high quality MPs essential for structural and functional analysis. Functional synthesis of MPs in vivo is challenging due to low yields, solubilization and purification problems, and overexpression often leads to toxicity. Having a flexible approach and faster synthesis method are crucial for synthesizing a wide range of high quality MPs which might help the researchers and drug companies to develop functional assays and to design new therapeutics. For more detailed understanding of the protein function, one needs to have an open isolated system where one can vary the parameters regulating the protein expression
systematically. Cell-free system offers all the conveniences required for proper synthesis of MPs. This method offers a high degree of controllability and provides a completely open system allowing direct manipulation of the reaction conditions to optimize protein folding, disulfide bond
formation, incorporation of noncanonical amino acids and the synthesis of toxic proteins [4,5,6,7,8,9]. In comparison to conventional cell-based systems
, cell-free systems offer rapid protein synthesis, purification and functional analysis. One of the most widely used cell-free systems is based on E. coli extracts. This system is widely used for synthesis of MPs in the presence of membrane solubilization supplements in the form of nanodiscs
, detergents, proteoliposomes
etc. added externally into the cell-free reaction [5, 10]. Nanodiscs
are synthetic discoidal nanoparticles consisting of a phospholipid bilayer
surrounded by two copies of membrane scaffold proteins (MSPs). MSPs are modified apolipoproteins consisting of a hydrophobic part toward the lipid bilayer
and a hydrophilic part outside thus providing stability
to the nanodiscs
and make them soluble without any detergents [8]. These nanoparticles can be added directly into the cell-free reaction system. Another existing cell-free system derived from insect (Spodoptera frugiperda Sf21) extracts is also used for synthesizing MPs. This eukaryotic cell-free system offers additional advantages in the form of native, ER-derived endogenous microsomes. Such microsomes contain the entire translocon machinery responsible for proper folding of MPs [8,9,10,11,12,13]. Recently, the potassium channel
KcsA was synthesized successfully in this system [13]. The synthesized MP showed tetrameric configuration and exhibited single-channel activity characteristic to the protein.
In this chapter, we will present a general method for measuring the
functionality of MPs derived from E. coli and insect-based cell-free systems. The expression and analysis will be shown exemplary with the proteins
bacteriorhodopsin (BR) and mannitol permease (MtlA)
using the E. coli cell-free system (Subheading 3.2), whereas the insect cell-free system will be used to produce the potassium channel protein KcsA (Subheading 3.3). We recommend using these proteins as positive controls when establishing the described protocols for your proteins of interest. The methodology we present here is also suitable for functional analysis of MPs synthesized by additional cell-free systems not discussed in this chapter. The main objective of this chapter is to propose two simple methods for the functional analysis of MPs derived from cell-free systems. These protocols can also be applied for screening protein variants.