Researchers at UC Irvine have developed a cell-free (CF) protein synthesis system to solubilize and synthesize highly hydrophobic membrane proteins that would typically aggregate using current CF synthesis systems. With such high amounts of synthesized proteins, researchers intend to build protein microarrays for diagnostic purposes.
Cell-free protein expression systems have been in use for several years to express and synthesize proteins in vitro. This technique is faster than other methods as it doesn’t require extensive cell culture and protein purification. However, membrane proteins that have hydrophobic surfaces and a low level of expression, form aggregates and are typically refolded into functional proteins. To address the problems associated with refolding membrane proteins, an alternative for proper synthesis using CF systems is required.
To overcome this problem, researchers at UC Irvine have developed a CF protein synthesis system where they demonstrate an aggregation free synthesis of membrane proteins. This method involves an addition of mild detergent, such as Brjj-78 or Brjj-98, to the reaction mixture. Detergent addition directly yields properly folded proteins in the solution. Researchers have demonstrated their method by successfully synthesizing solubilized amounts of up to 52 variants of different insoluble membrane proteins across three organisms. Such larger amounts of proteins can be printed on protein arrays, which would result in improvement of recognition by antibodies when probed with patient sera.
This approach eliminates the problem of protein aggregation in CF systems and results in properly folded membrane proteins that could be used for protein microarrays. Furthermore, it produces similar amounts of membrane proteins to commercially available kits, and can be performed in high throughput format.
This method has been currently optimized for various insoluble proteins across three different organisms.