Membrane proteins, such as receptors, channels, and transporters, play critical roles in a wide variety of biological processes. Because of their highly hydrophobic and intricate structures, one bottleneck in studying membrane proteins is the difficulty expressing them in sufficient quantities as properly folded and stable proteins. Often multiple rounds of protein engineering are required to optimize the process, which could take a year or longer to perfect.
“Many researchers use the baculovirus-based expression system because, unlike bacterial systems, it adds appropriate post-translational modifications required for proper folding and function,” says Hao Chen, Ph.D., senior scientist, protein technologies, Amgen.
Dr. Chen and his team developed a method for expressing proteins in baculovirus that modifies the traditional method to dramatically reduce time and resources. “Baculovirus manipulations generally require creating, titering, and amplifying viral stocks that are used to express proteins in insect cells,” notes Dr Chen. “This is a laborious process that takes about 3–4 weeks. We developed a method to screen membrane protein expression that typically takes only 2–3 days.”
The team made expression plasmids that fused green fluorescent protein (GFP) to the target membrane proteins and transfected the construct directly into insect cells without going through typical viral approaches.
“Transient expression of recombinant proteins in insect cells has not been widely adopted due to its low protein yield and difficulty scaling up. However, by fusing GFP to the membrane protein, we can directly monitor the resulting fusion proteins in whole cells for their subcellular localizations using fluorescence microscopy,” continued Dr. Chen. “Additionally, we use a method called fluorescence-detection size exclusion chromatography, or FSEC, that requires only nanogram levels of unpurified protein to characterize expression level and approximate molecular size and stability of the over-expressed integral membrane proteins.”
Dr. Chen utilized the ion-channel protein ASIC3 and transporter SLC7A5 to demonstrate the technique. “If a researcher has an HPLC capability, this technique is super easy and ultrasensitive,” asserts Dr. Chen.
The challenge of the technique is its limited throughput. Still, according to Dr. Chen, many people have shown great interest in the method because it provides a way to “tackle difficult or poorly characterized proteins, the number of which continues to grow.”