Portland, Ore. - Using the bioengineering technique of gene splicing, Johns Hopkins University researchers have built an artificial protein with unique switching behavior. The result is a biomolecule in which one segment acts as a control that activates another segment. This line of research could lead to highly selective drug delivery systems or handheld devices that would signal the presence of biowarfare agents, said Johns Hopkins professor Marc Ostermeier.
"We take two proteins with different functions and fuse them in such a way as to form a switch," said Ostermeier, who was assisted by doctoral student Gurkan Guntas. Both proteins occur naturally in the E. coli bacteria but perform different functions. One, beta-lactamase, counters the action of penicillin and the other-maltose-binds sugar molecules to provide energy for the bacteria. Normally the two proteins operate in separate areas of cell metabolism, but with the gene-splicing technique, the researchers were able to bond them together to create a novel operation. The maltose-binding operation is a trigger for the immune response to antibiotics.
In the Johns Hopkins scheme, the protein that recognizes a biological agent does not have to provide the signal to the outside world-an advantage for biosensor design. Instead, it is fused with a protein that does produce an easily detectable signal. "You are free to choose a signal-transducing protein that will give you the best signal," said Ostermeier, who presented his work last month at the 225th annual meeting of the American Chemical Society. Using the technique, a sensor could be designed to be sensitive only to cancer cells, for example, activating the second part of the protein to deliver a lethal drug to them.
The basic process comes from the emerging field of proteomics-the study of how the information in DNA is used to construct proteins. Ostermeier is working with a technique called "domain insertion," splicing together protein sequences with different functions. His sensing proteins exhibit shape-changing, or allosteric, transformations that turn on a molecular switch in the second, signaling protein.
Such molecular switches could be applied as supersensitive sensors that detect even a single molecule of a biological warfare agent. Medically, molecular switches have even wider-scale potential in early detection of hard-to-diagnosis maladies. The recent outbreak and rapid spread of the SARS virus provides a good example of how a biomolecule detector could aid health workers. Even though the DNA of the coronavirus was sequenced in a record six days by a team of medical researchers at the British Columbia Cancer Agency in Vancouver, that in itself will not stop the spread of the disease in an era of rapid, worldwide travel. However, a handheld lab-on-a-chip using Ostermeier's engineered protein would make it possible to isolate people who have the disease before they board an airplane.
Ostermeier's laboratory studies how to link molecular-level signals such as ligand binding, protein-protein interactions or pH changes to switches signaling macro-level alerts, such as fluorescence. For instance, a sensor could fluoresce green when sensing a toxin.
The technique uses wet chemistry to splice two disparate enzymes-one to sense and one to signal-so that "turning on" the sensing protein will trigger the signaling protein. In Ostermeier's experiment, a maltose-binding protein found in a harmless form of E. coli bacteria was spliced with a beta-lactamase enzyme, which controls the bacteria's immune system. The presence of maltose switched on the beta-lactamase, rendering the bacteria immune. "Of the two proteins, one binds to the molecule to be recognized, resulting in a conformational change that is transmitted to the other protein acting as the switch," said Ostermeier.
The hallmarks of the technique are this use of allosteric mechanisms plus the harnessing of evolutionary principles to select from among a combinatorial library of candidates. The creed at Ostermeier's lab is that when knowledge fails, build a library of candidates and test for the "good" ones.
For the current experiment, Ostermeier went back to the genes from which the maltose and beta-lactamase proteins are produced. In a beaker full of maltose-binding genes, he poured enzymes that snipped them into severed halves. A second enzyme then reattached the severed ends to each side of a beta-lactamase gene. Each gene in the resulting collage of cut-and-pasted genes produced a single protein strand that combined the proteins of maltose and beta-lactamase under the direction of the spliced genes.
To verify that he had produced switchable proteins, Ostermeier mixed the E. coli bacteria in a solution of maltose, antibiotics and his engineered proteins. In theory the maltose would stimulate the artificial protein to activate the bacteria's immune system. Thus, a high survival rate for the bacteria in the presence of anitbiotics would indicate effective molecular switches.
In general, molecular switches represent one the smallest possible "physical" representations of a bit-on or off. They are so small, in fact, that they are usually used with overwhelm-ing redundancy. For instance, a badge sensor would have a dense layer of enzymes on its surface so that their combined fluorescing could be easily seen by the naked eye. On the molecular level, an allosteric change of shape transmits the "on" signal via fluorescence.
Ostermeier hopes to pioneer molecular switches by harnessing the evolutionary principle of natural selection to find just the right combination of two proteins, thereby enabling highly refined functionality that would be impossible to create from scratch with current bioengineering techniques. His strategy is to insert one strand "into" the other. The exact insertion spot determines the resulting shape, but it is impossibly complex to calculate the outcome ahead of time even for the fastest supercomputers. Instead, Ostermeier uses the natural selection of directed evolution.
"To get them to talk to one another, you want to fuse the two proteins, but if you fuse them end to end, then you only have a single connection point," he said. "So the idea was to insert one into the other so you have two connection points, for more reliable operation. This way, right from the start you have a much higher chance that when the first protein binds to something, the second one is going to feel it and turn on too."
Ostermeier picked the middle of one protein as the insertion point. "If you start from one end you have the start of one protein, then all of the second protein and then the last half of the first protein," he said.
Today, knowledge of protein folding is scanty. The impossibly complex shapes into which long strands of proteins can morph are not well enough understood to decide the optimal place to make such an insertion. Without a usable algorithm, researchers like Ostermeier turn to directed evolution in a test tube-by creating a test tube full of every possible way of doing the splice then testing to find a winner.
"We used a molecular-biology technique to make a test tube full of every possible way of inserting one protein into the other, then [we] use directed evolution in the lab to select for those that worked best," said Ostermeier.
Ostermeier believes this technology will develop from a "proof-of-concept" status to a higher-performance model within the next year. Within five years, he said, some real applications, like the sensor badge, will be tackled.
"From here, we want to make better switches-and we have ideas about how to do that," said Ostermeier. "Currently we increase activity about 100 percent in our signal protein, but we would like to have a much larger effect-the ideal goal would be no activity [in the absence of anything to sense] or full activity for a true on-or-off switch."
A second goal is to study the best-performing examples selected by directed evolution in order to discover their potency. Why do some shape changes in proteins work better than others in making the second protein turn on?
Some of the fastest supercomputers in the world are at work on the subject, but Ostermeier intends to inspect the real-world examples of "good" proteins to try to discern a pattern.
Ostermeier is also keen to move the technology out of the lab. "I would like to make a molecular sensor next," he said. "We also want to better understand the switches we already have, because it would be much more satisfying if we could design these switches instead of just using the evolutionary approach."
Johns Hopkins University (Baltimore) has applied for several patents on Ostermeier's molecular switch. The research was funded by the American Cancer Society as well as the Maryland Cigarette Restitution Fund.