Screening for critical drug targets known as G-protein-coupled receptors (GPCRs) is now possible without the need to extract these proteins from their native cells. Extraction requires the use of stabilizing lipids, which damage the structural integrity and functionality of GPCRs. The cell-free approach was developed by Madhavan Nallani from the A*STAR Institute of Materials Research and Engineering, Singapore, along with co-workers in Singapore, Germany and Austria.
Nallani and co-workers focused on GPCRs because they are ideal drug targets that pass through cell membranes. These proteins are involved in cell communication, cell adhesion and signal transduction, as well as major illnesses, including hypertension and diabetes.
The team’s approach produced GPCRs directly in artificial cell-like pockets called polymersomes. “This [approach] circumvents the tedious protein isolation process and the use of lipids or detergents as stabilizing agents,” says Nallani.
As a proof of concept, the researchers synthesized the GPCR dopamine receptor D2 in vitro in the presence of polymersomes and the DNA that encodes for the protein. “This cell-free expression provides an easy way to produce proteins directly from their DNA,” notes Nallani. The proteins generated by transcription and translation became incorporated into the polymer-based membranes via spontaneous self-assembly.
Characterization using fluorescently labeled antibodies that are receptor-specific showed that the self-assembled product displayed stronger fluorescence than the unmodified polymersomes, confirming the insertion of the receptor. This characterization also indicated that the membrane-incorporated portion of the receptor was properly oriented in the polymersomes.
Nallani’s team further investigated the structure of the polymersome-inserted receptor using a fluorescently tagged dopamine. “Dopamine will bind only if the receptor is correctly folded and oriented,” explains Nallani. After incubation, the GPCR-modified polymersomes showed higher fluorescence than the negative controls, indicating that the receptor maintained its structural integrity upon insertion into the polymersome.
The researchers assessed the potential use of the GPCR-modified polymersomes for drug screening and biosensing by synthesizing the receptor in the presence of polymersomes, which they patterned onto glass surfaces. Incubation of the resulting material with fluorescently labeled dopamine illuminated the patterns — proof of a successful GPCR addition. Exposure of these fluorescent patterns to increasing concentrations of unlabeled dopamine caused the fluorescence to decrease, showing that dopamine displaced the fluorescent ligand.
“We are developing this technology through a commercialization project from the A*STAR Exploit Technologies Pte Ltd and focusing on small-molecule and antibody screening,” says Nallani. His team is planning to form a start-up company with this approach within the next few months.
The A*STAR-affiliated researchers contributing to this research are from the Institute of Materials Research and Engineering
May, S., Andreasson-Ochsner, M., Fu, Z., Low, Y. X., Tan, D. et al. In vitro expressed GPCR inserted in polymersome membranes for ligand-binding studies. Angewandte Chemie International Edition 52, 749–753 (2013).
Water forms 'spine of hydration' around DNA, group finds
26.05.2017 | Cornell University
How herpesviruses win the footrace against the immune system
26.05.2017 | Helmholtz-Zentrum für Infektionsforschung
Staphylococcus aureus is a feared pathogen (MRSA, multi-resistant S. aureus) due to frequent resistances against many antibiotics, especially in hospital infections. Researchers at the Paul-Ehrlich-Institut have identified immunological processes that prevent a successful immune response directed against the pathogenic agent. The delivery of bacterial proteins with RNA adjuvant or messenger RNA (mRNA) into immune cells allows the re-direction of the immune response towards an active defense against S. aureus. This could be of significant importance for the development of an effective vaccine. PLOS Pathogens has published these research results online on 25 May 2017.
Staphylococcus aureus (S. aureus) is a bacterium that colonizes by far more than half of the skin and the mucosa of adults, usually without causing infections....
Physicists from the University of Würzburg are capable of generating identical looking single light particles at the push of a button. Two new studies now demonstrate the potential this method holds.
The quantum computer has fuelled the imagination of scientists for decades: It is based on fundamentally different phenomena than a conventional computer....
An international team of physicists has monitored the scattering behaviour of electrons in a non-conducting material in real-time. Their insights could be beneficial for radiotherapy.
We can refer to electrons in non-conducting materials as ‘sluggish’. Typically, they remain fixed in a location, deep inside an atomic composite. It is hence...
Two-dimensional magnetic structures are regarded as a promising material for new types of data storage, since the magnetic properties of individual molecular building blocks can be investigated and modified. For the first time, researchers have now produced a wafer-thin ferrimagnet, in which molecules with different magnetic centers arrange themselves on a gold surface to form a checkerboard pattern. Scientists at the Swiss Nanoscience Institute at the University of Basel and the Paul Scherrer Institute published their findings in the journal Nature Communications.
Ferrimagnets are composed of two centers which are magnetized at different strengths and point in opposing directions. Two-dimensional, quasi-flat ferrimagnets...
An Australian-Chinese research team has created the world's thinnest hologram, paving the way towards the integration of 3D holography into everyday...
24.05.2017 | Event News
23.05.2017 | Event News
22.05.2017 | Event News
26.05.2017 | Life Sciences
26.05.2017 | Life Sciences
26.05.2017 | Physics and Astronomy