Skip to main content
Premium Trial:

Request an Annual Quote

Oxford Researchers Develop Time-Saving Technique For Putting Membrane Proteins Into Lipid Bilayers


Using a glass rod with a sharp tip, University of Oxford researchers have developed what they consider to be a time-saving technique for putting membrane proteins into artificial membranes.

The researchers use the rod, which has a tip that is about 50 microns in diameter, to touch a colony of E. coli bacteria that overexpress the membrane protein of interest. They then touch the same rod to an artificial lipid bilayer membrane. The membrane protein transfers from the bacterial colony into the membrane.

"It works every time," said Matthew Holden, a postdoc in Hagan Bayley's laboratory at the University of Oxford who led the development of the technique, which was published this week in the online version of Nature Chemical Biology. "You just smash the tip right into the membrane."

Most researchers express their proteins of interest within E. coli cells, purify the proteins and run them out on a gel, cut out the gel band of interest, extract out the proteins from the gel, add the proteins in solution to a solution with the bilayer, and hope that the proteins go into the bilayer by themselves.

"This cuts out the protein purification altogether," said Holden. "You get the DNA, the bug amplifies the DNA, you plate the cells out, let them grow, and do the transfer."

"I believe it is a significant breakthrough for the field of chemical-biology that investigates individual membrane proteins and their interactions with the membrane bilayer and ligands, as well as protein-protein interactions."

Though the technique appears to be simple and intuitive, it has hardly been used, probably because researchers thought the bilayers would break if a solid object was bashed into them, said Holden.

"People just started off with the presupposition that the bilayers would break," said Holden. "If anyone just looked under the microscope, they would see that that isn't true."

In addition to saving time, the direct transfer method enables researchers to insert proteins that are difficult to purify into membranes.

"Sometimes you work with constructs that are difficult to purify, or to refold," said Holden. "This [method] is going to be of great interest to look at proteins that we can't purify, but that we can express."

The new method can even be used to insert proteins that are comprised of more than one subunit, Holden noted. He and his colleagues showed that the toxin leukocidin, an octomeric protein composed of LukF and LukS subunits, could be easily transferred to a planar bilayer by first expressing LukF and LukS in different E. coli colonies, mixing together the two colonies, and then doing the direct transfer using the glass rod.

"We simply stuck the probe in the colonies which were squished together and the monomers self-assembled and we got a functional pore," said Holden.

Holden said in the future, he might try using the technique to transfer even more complex proteins made out of more than two subunits. He is also considering expressing and transferring eukaryotic membrane proteins using the new technique.

"There's loads of possibilities," said Holden. "You can look at a lot of membrane proteins without wasting a tremendous amount of time. One idea is to use the method to build membrane protein arrays — microchips covered with little membranes with little pores each designed to detect a certain drug."

In addition to transferring leukocidin, the researchers also transferred potassium channels, hemolysin, and KcsA channels into lipid bilayers using the method.

Holden said he and his colleagues filed a patent for their method in March 2005. However, they "don't expect to make money out of it," said Holden.

So far, the researchers have avoided using the method to try to insert membrane proteins into live cells, Holden said.

"That's not the line of research we're interested in," he said. "We're looking at protein function on a single molecule level."

In addition, it would be difficult to apply the technique to live cells because cells are generally smaller than the size of the glass probe, and it would be difficult to control the movement of the cell, said Holden.

"I guess you could impale a giant oocyte, but the idea is that live cells already have so many pores for exchanging ions, building voltage and pH gradients, taking in food, secreting stuff," he said. "This technique is really geared toward the planar bilayer. We're using the approach for electrical recording."

Josip Blonder, a senior research scientist who works with membrane proteins in the Laboratory of Proteomics and Analytical Technologies at the National Cancer Institute at Frederick (see ProteoMonitor 6/10/2005), said that he does not foresee large-scale utility of the new technique in traditional mass spec-based proteomics where complex membrane protein mixtures are studied. However, he said the method could be used for targeted investigations of a particular membrane protein or membrane protein complex.

"This approach is time saving and less laborious when compared to classical techniques that rely on in vitro transcription and translation and various protein purification protocols," said Blonder. "I believe it is a significant breakthrough for the field of chemical biology that investigates individual membrane proteins and their interactions with the membrane bilayer and ligands, as well as protein-protein interactions."

— Tien-Shun Lee ([email protected])

The Scan

Transcriptomic, Epigenetic Study Appears to Explain Anti-Viral Effects of TB Vaccine

Researchers report in Science Advances on an interferon signature and long-term shifts in monocyte cell DNA methylation in Bacille Calmette-Guérin-vaccinated infant samples.

DNA Storage Method Taps Into Gene Editing Technology

With a dual-plasmid system informed by gene editing, researchers re-wrote DNA sequences in E. coli to store Charles Dickens prose over hundreds of generations, as they recount in Science Advances.

Researchers Model Microbiome Dynamics in Effort to Understand Chronic Human Conditions

Investigators demonstrate in PLOS Computational Biology a computational method for following microbiome dynamics in the absence of longitudinally collected samples.

New Study Highlights Role of Genetics in ADHD

Researchers report in Nature Genetics on differences in genetic architecture between ADHD affecting children versus ADHD that persists into adulthood or is diagnosed in adults.