Genetic code used to engineer a living protein

Yale University researchers have successfully re-engineered the protein-making machinery in bacteria, a technical tour de force that promises to revolutionize the study and treatment of a variety of diseases.

"Essentially, we have expanded the genetic code of E. coli, which allows us synthesize special forms of proteins that can mimic natural or disease states," said Jesse Rinehart of the Department of Cellular and Molecular Physiology and co-corresponding author of the research published in the August 26 issue of the journal Science.

Since the structure of DNA was revealed in the 1950s, scientists have been working hard to understand the nature of the genetic code. Decades of research and recent advances in the field of synthetic biology have given researchers the tools to modify the natural genetic code within organisms and even rewrite the universal recipe for life.

"What we have done is taken synthetic biology and turned it around to give us real biology that has been synthesized," Rinehart explained.

The Yale team — under the direction of Dieter Söll, Sterling Professor of Molecular Biophysics and Biochemistry, professor of chemistry and corresponding author of the paper — developed a new way to influence the behavior of proteins, which carry out almost all of life's functions. Instead of creating something new in nature, the researchers essentially induced phosphorylation, a fundamental process that occurs in all forms of life and can dramatically change a protein's function. The rules for protein phosphorylation are not directly coded in the DNA but instead occur after the protein is made. The Yale researchers fundamentally rewrote these rules by expanding the E. coli genetic code to include phosphoserine, and for the first time directed protein phosphorylation via DNA.

This new technology now enables the production of human proteins with their naturally occurring phosphorylation sites, a state crucial to understanding disease processes. Previously, scientists lacked the ability to study proteins in their phosphorylated or active state. This has hindered research in diseases such as cancer, which is marked by damagingly high levels of protein activation.

"What we are doing is playing with biological switches — turning proteins on or off — which will give us a completely new way to study disease states and hopefully guide the discovery of new drugs," Rinehart said.

"We had to give some very ancient proteins a few modern upgrades," Söll said.

Söll and Rinehart now are attempting to create proteins in states known to be linked to cancer, type 2 diabetes, and hypertension. Both men, however, stressed the technique can be done for any type of protein.

"Dr. Söll and his colleagues have provided researchers with a powerful new tool to use in uncovering how cells regulate a broad range of processes, including cell division, differentiation and metabolism," said Michael Bender, who oversees protein synthesis grants at the National Institute of General Medical Sciences of the National Institutes of Health.

Provided by Yale University (news : web)

Scientists engineer nanoscale vaults to encapsulate 'nanodisks' for drug delivery

There's no question, drugs work in treating disease. But can they work better, and safer? In recent years, researchers have grappled with the challenge of administering therapeutics in a way that boosts their effectiveness by targeting specific cells in the body while minimizing their potential damage to healthy tissue.

The development of new methods that use engineered nanomaterials to transport drugs and release them directly into cells holds great potential in this area. And while several such drug-delivery systems -- including some that use dendrimers, liposomes or polyethylene glycol -- have won approval for clinical use, they have been hampered by size limitations and ineffectiveness in accurately targeting tissues.

Now, researchers at UCLA have developed a new and potentially far more effective means of targeted drug delivery using nanotechnology.

In a study to be published in the May 23 print issue of the journal Small, they demonstrate the ability to package drug-loaded "nanodisks" into vault nanoparticles, naturally occurring nanoscale capsules that have been engineered for therapeutic drug delivery. The study represents the first example of using vaults toward this goal.

The UCLA research team was led by Leonard H. Rome and included his colleagues Daniel C. Buehler and Valerie Kickhoefer from the UCLA Department of Biological Chemistry; Daniel B. Toso and Z. Hong Zhou from the UCLA Department of Microbiology, Immunology and Molecular Genetics; and the California NanoSystems Institute (CNSI) at UCLA.

Vault nanoparticles are found in the cytoplasm of all mammalian cells and are one of the largest known ribonucleoprotein complexes in the sub-100-nanometer range. A vault is essentially barrel-shaped nanocapsule with a large, hollow interior -- properties that make them ripe for engineering into a drug-delivery vehicles. The ability to encapsulate small-molecule therapeutic compounds into vaults is critical to their development for drug delivery.

Recombinant vaults are nonimmunogenic and have undergone significant engineering, including cell-surface receptor targeting and the encapsulation of a wide variety of proteins.

"A vault is a naturally occurring protein particle and so it causes no harm to the body," said Rome, CNSI associate director and a professor of biological chemistry. "These vaults release therapeutics slowly, like a strainer, through tiny, tiny holes, which provides great flexibility for drug delivery."

The internal cavity of the recombinant vault nanoparticle is large enough to hold hundreds of drugs, and because vaults are the size of small microbes, a vault particle containing drugs can easily be taken up into targeted cells.

With the goal of creating a vault capable of encapsulating therapeutic compounds for drug delivery, UCLA doctoral student Daniel Buhler designed a strategy to package another nanoparticle, known as a nanodisk (ND), into the vault's inner cavity, or lumen.

"By packaging drug-loaded NDs into the vault lumen, the ND and its contents would be shielded from the external medium," Buehler said. "Moreover, given the large vault interior, it is conceivable that multiple NDs could be packaged, which would considerably increase the localized drug concentration."

According to researcher Zhou, a professor of microbiology, immunology and molecular genetics and director of the CNSI's Electron Imaging Center for NanoMachines, electron microscopy and X-ray crystallography studies have revealed that both endogenous and recombinant vaults have a thin protein shell enclosing a large internal volume of about 100,000 cubic nanometers, which could potentially hold hundreds to thousands of small-molecular-weight compounds.

"These features make recombinant vaults an attractive target for engineering as a platform for drug delivery," Zhou said. "Our study represents the first example of using vaults toward this goal."

"Vaults can have a broad nanosystems application as malleable nanocapsules," Rome added.

The recombinant vaults are engineered to encapsulate the highly insoluble and toxic hydrophobic compound all-trans retinoic acid (ATRA) using a vault-binding lipoprotein complex that forms a lipid bilayer nanodisk.

The research was supported by the UC Discovery Grant Program, in collaboration with the research team's corporate sponsor, Abraxis Biosciences Inc., and by the Mather's Charitable Foundation and an NIH/NIBIB Award.

Story Source:

The above story is reprinted (with editorial adaptation) from materials provided by University of California - Los Angeles.

Journal Reference:

Daniel C. Buehler, Daniel B. Toso, Valerie A. Kickhoefer, Z. Hong Zhou, Leonard H. Rome. Vaults Engineered for Hydrophobic Drug Delivery. Small, 2011; DOI: 10.1002/smll.201002274