Membrane proteins perform a broad range of essential functions and their dysregulation has been linked to conditions spanning cystic fibrosis, diabetes, and rheumatoid arthritis to Alzheimer’s disease, heart failure, and cancer. As a consequence, membrane proteins now constitute around 60% of known and novel drug targets. Yet despite their critical importance, membrane proteins have long been considered difficult to characterize due to the intrinsic challenges of isolating them from native sources. Various solutions have been developed to support membrane protein research, including advanced production methods based on recombinant protein expression technology.
Membrane proteins, as the name suggests, are proteins that form part of, or interact with, a biological membrane. They are categorized into two main groups on the basis of membrane association. The first group, known as the integral membrane proteins, are permanent membrane constituents that serve as receptors, transduce signals between a cell’s internal and external environments, and facilitate cellular adhesion, as well as perform countless other functions. The second group, referred to as the peripheral membrane proteins, associate only transiently with the membrane, either via interaction with integral membrane proteins or by adhering directly to the lipid layer. Well-known examples of membrane proteins include the G-protein coupled receptors (GPCRs), the claudins, and many of the cluster of differentiation (CD) antigens.
A main challenge faced by researchers when isolating proteins from biological membranes lies in obtaining a product of sufficient yield and quality. This is because membrane proteins are often expressed at only low levels, while many common isolation techniques are inherently variable and risk damaging the target of interest. Notably, integral membrane proteins can be especially difficult to obtain as they typically require more stringent isolation methods than their peripheral counterparts. One way of addressing these issues is to use an optimized detergent technology platform for solubilizing and extracting integral membrane proteins while retaining both structure and physical properties. Another approach, known as nanodisc technology, uses specialized synthetic polymers for membrane protein isolation, purification, and characterization.
Recombinant protein expression technology has long been used as an alternative to native protein isolation. It involves introducing a gene encoding the protein of interest into a host cell via a vector, before expanding the host in culture and extracting the protein product. Over the years, methods have evolved from using double-stranded DNA plasmid vectors and bacterial hosts (predominantly E. coli) to using viral vectors and mammalian hosts such as human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells. These advances have provided proteins that better resemble the native state in terms of both structure and function.
One way in which recombinant protein expression technology has been successfully applied for membrane protein research is through a combination of enveloped virus-like particle (eVLP) technology platforms with HEK293 expression hosts. This approach allows for a full-length target membrane protein to be expressed on the VLP surface and detected as if it were a native protein, as shown in Figure 1. As well as generating protein products that are well suited for immunization and antibody screening purposes, a further advantage of this method is that producing microgram amounts of protein takes as little as 2–3 weeks from initial vector construction.
Figure 1. Principles of VLP-based protein expression
There are an almost limitless number of ways in which recombinant membrane proteins can be applied for scientific research. Examples include their use as qualitative controls for western blot, where they provide a positive readout against which test samples can be compared, and their utility as quantitative controls for ELISA, where they can form the basis of a standard curve. Recombinant membrane proteins are also useful for many other types of studies, including those designed to measure antibody or small molecule binding kinetics, detect chimeric antigen receptors (CARs), or investigate the formation of large multi-protein complexes.
Sino Biological focuses on the development of multi-pass transmembrane proteins and has successfully established three platforms for their production. To learn more, please visit sinobiological.com