New Designer Proteins Come with Built-In Safeguards

By merging two genes into a single DNA sequence, CUIMC synthetic biologists have created a method that could prevent human-engineered proteins from spreading into the wild, as well as stabilize synthetic proteins so they don’t change over time.

Background

Protein engineering is a relatively young field that creates new proteins never seen before in nature. Today’s protein engineers usually create synthetic proteins by making small changes to the gene that encodes a naturally occurring protein. The variety of synthetic proteins range from stain-removing enzymes that have improved detergents to a long-acting insulin that’s used by millions of people with diabetes.

But two big unsolved challenges for protein engineers remain: The gene encoding the synthetic protein needs to be contained to prevent escape into other organisms and the gene needs to resist mutating over time so the protein doesn’t lose its function.

Synthetic biologists at Columbia University Vagelos College of Physicians and Surgeons have developed a method, now published in the journal Science, that can address both issues.

Inspiration from viruses

In devising the method, the researchers were inspired by overlapping genes in viruses. When two different genes overlap, they occupy the same sequence of DNA. But the genes are read in different frames so that two different proteins are produced.

In overlapping genes, a random mutation in the sequence may not affect one gene, but it’s likely that it will harm the second gene.

“Overlapping genes essentially lock in a specific DNA sequence, and we thought we could exploit this idea for synthetic biology,” says Harris Wang, PhD, assistant professor of systems biology, who developed the new method with graduate student Tomasz Blazejewski and postdoctoral scientist Hsing-I Ho, PhD.

Computer algorithm entangles genes

The CAMEOS technique developed by Wang, Blazejewski, and Ho creates a single DNA sequence containing two genes that encode two separate proteins.

A computer algorithm in CAMEOS starts with the genetic code of two natural genes and devises ways to combine the two genes into a single DNA sequence. 

To accomplish such entanglement, bases in each gene need to be altered but without altering the function of the gene’s protein. CAMEOS taps a database of hundreds of thousands of gene sequences to determine which base changes are likely to succeed and which are likely to fail.

The final predicted sequences are then printed and tested inside living cells using high throughput techniques that make possible the testing of thousands of different sequences in a short period of time.

“Ten years ago, we didn’t have the technology that would make this possible,” Wang says. “We didn’t have enough sequences in the database to make informed predictions and we didn’t have a way to synthesize long DNA sequences for testing our predictions.”

Biocontainment and gene stability

To prevent a synthetic gene from escaping into the wild, the Columbia researchers used CAMEOS to entangle it with a gene that produces a toxic protein.

When inserted into bacterial cells engineered to make the antidote, the entangled genes produce the synthetic gene and the toxin. Other bacteria could take up the entangled gene, but that meant instant death once the toxin was created. 

Similar designs “could be useful for agricultural purposes where you don’t want a synthetic gene to spread to natural crops,” Blazejewski says, “or in any situation when you don’t want your synthetic DNA to escape from the lab.”

By locking in a DNA sequence, gene entanglement also stabilizes engineered genes and prevents the synthetic protein from losing its functions (or acquiring unwanted ones).

“Instability is an issue now in industries that use vats of cells to produce engineered proteins,” Ho says. “The reaction will only run for a certain amount of time before mutations take over. With CAMEOS, it may be possible to sustain the reaction for longer.”

The U.S. Department of Defense, which is interested in increasing the stability of engineered proteins, helped fund the development of CAMEOS. Incorporated into microbes, engineered proteins could help protect equipment against corrosion, warn soldiers of the presence of chemical weapons, or produce fuel or drugs on demand.