Using a lab technique called domain insertion, Johns Hopkins researchers have joined two proteins in a way that creates a molecular "switch." The result, the researchers say, is a microscopic protein partnership in which one member controls the activity of the other. Similarly coupled proteins may someday be used to produce specialized molecules that deliver lethal drugs only to cancerous cells. They also might be used to set off a warning signal when biological warfare agents are present.
The technique used to produce this molecular switch was reported March 27 in New Orleans at the 225th national meeting of the American Chemical Society.
"We've taken two proteins that normally have nothing to do with one another, spliced them together genetically and created a fusion protein in which the two components now 'talk' to one another," said Marc Ostermeier, assistant professor in the Department of Chemical and Biomolecular Engineering in the Whiting School. "More important, we've shown that one of these partners is able to modulate or control the activity of the other. This could lead to very exciting practical applications in medical treatment and bio-sensing."
To prove the production of a molecular switch is possible, Ostermeier, assisted by doctoral student Gurkan Guntas, started with two proteins that typically do not interact: beta-lactamase and the maltose binding protein found in a harmless form of E. coli bacteria. Each protein has a distinct activity that makes it easy to monitor. Beta-lactamase is an enzyme that can disable and degrade penicillinlike antibiotics. Maltose binding protein binds to a type of sugar called maltose that the E. coli cells can use as food.
Using a technique called domain insertion, the Johns Hopkins researchers placed beta-lactamase genes inside genes for maltose binding protein. To do this, they snipped the maltose binding genes, using enzymes that act like molecular scissors to cut the genes as though they were tiny strips of paper. A second enzyme was used to re-attach these severed strips to each side of a beta-lactamase gene, producing a single gene strip measuring approximately the combined length of the original pieces. This random cut-and-paste process took place within a test tube and created hundreds of thousands of combined genes. Because the pieces were cut and reassembled at different locations along the maltose binding gene, the combined genes produced new proteins with different characteristics.
Ostermeier believed a very small number of these new fusion proteins might possess the molecular switch behavior he was looking for. To find them, he and Guntas took a cue from the process of evolution, or "survival of the fittest." By looking for the E. coli that thrived in maltose, they could isolate only the ones in which the maltose binding partner was still active (in other words, it still bound itself to maltose). By then mixing them with an antibiotic, the researchers could find the ones in which the beta-lactamase remained active and capable of reacting against the antibiotic. Through such survival tests, the researchers ultimately were able to find two fusion proteins in which not only were both proteins still active but in which the presence of maltose actually caused the beta-lactamase partner to step up its attack on an antibiotic.
"In other words," Ostermeier said, "one part of this coupled protein sent a signal, telling the other part to change its behavior. This is the first clear demonstration that you can apply the domain insertion technique to control the activity of an enzyme. If we can replicate this with other proteins, we can create biological agents that don't exist in nature but can be very useful in important applications."
For example, Ostermeier said, one part of a fusion protein might react only to cancer cells, signaling its partner to release a toxin to kill the diseased tissue. Healthy cells, however, would not set off the switch and would thus be left unharmed. Ostermeier also suggested that one part of a fusion protein might react to the presence of a biological warfare agent, signaling its partner to set off a bright green fluorescent glow that could alert soldiers and others to the danger.
Johns Hopkins has applied for U.S. and international patents related to Ostermeier's molecular switch technology and the techniques used to produce them. Ostermeier's research has been funded by grants from the American Cancer Society and the Maryland Cigarette Restitution Fund.